Method of producing emulsions

ABSTRACT

Devices, systems, and their methods of use, for generating droplets are provided. One or more geometric parameters of a microfluidic channel can be selected to generate droplets of a desired and predictable droplet size.

BACKGROUND OF THE INVENTION

Many biomedical applications rely on high-throughput assays of samplescombined with one or more reagents in droplets. For example, in bothresearch and clinical applications, high-throughput genetic tests usingtarget-specific reagents are able to provide information about samplesin drug discovery, biomarker discovery, and clinical diagnostics, amongothers.

Improved devices and methods for producing droplets would be beneficial.

SUMMARY OF THE INVENTION

We have developed a microfluidic device that is capable of producingdroplets of a first liquid in a second liquid that is immiscible withthe first liquid.

In one aspect, the invention provides a device for producing droplets ofa first liquid in a second liquid. The device includes a channel and adroplet formation region configured to allow a liquid flowing from thechannel to expand in at least one dimension, e.g., having a shelfregion, a step region, or both.

In one embodiment, the device includes a) a first channel having a firstdepth, a first width, a first proximal end, and a first distal end; b) asecond channel having a second depth, a second width, a second proximalend, and a second distal end, where the second channel intersects thefirst channel between the first proximal and first distal ends; and c) adroplet formation region including a shelf region having a third depthand a third width, and a step region having a fourth depth, where theshelf region is configured to allow the first liquid to expand in atleast one dimension and has at least one inlet and at least one outlet,and where the shelf region is disposed between the first distal end andthe step region. The first channel and droplet formation region areconfigured to produce droplets of the first liquid in the second liquid.

In some embodiments, the first liquid contains particles. In certainembodiments, the first channel and the droplet formation region areconfigured to produce droplets including a single particle or a singleparticle of multiple types, e.g., one bead and one cell. In someembodiments, the third width increases from the inlet of the shelfregion to the outlet of the shelf region.

In certain embodiments, the device includes a first reservoir and asecond reservoir in fluid communication with the first proximal end andthe second proximal end, respectively. In further embodiments, thedevice includes a collection reservoir configured to collect dropletsformed in the droplet formation region. In certain embodiments, the stepregion and collection reservoir do not have orthogonal elements thatcontact the droplets when formed. In some embodiments, where the deviceis configured to produce a population of droplets that are substantiallystationary in the collection reservoir.

In some embodiments, the device includes a third channel having a thirdproximal end and a third distal end, where the third proximal end is influid communication with the shelf region and where the third distal endis in fluid communication with the step region (e.g., the third proximalend is fluidically connected to the shelf region and the third distalend is fluidically connected to the step region).

In further embodiments, the device includes a plurality of firstchannels, second channels, and droplet formation regions, e.g., that arefluidically independent to produce an array.

In a related aspect, the invention includes a system for producingdroplets of a first liquid in a second liquid, the system including a) adevice for producing droplets, where the device includes i) a firstchannel having a first depth, a first width, a first proximal end, and afirst distal end; ii) a second channel having a second depth, a secondwidth, a second proximal end, and a second distal end, where the secondchannel intersects the first channel between the first proximal andfirst distal ends; iii) a droplet formation region having a shelf regionhaving a third depth and a third width and a step region having a fourthdepth, where the shelf region is configured to allow the first liquid toexpand in at least one dimension and has at least one inlet and at leastone outlet, where the shelf region is disposed between the first distalend and the step region; iv) a first reservoir in fluid communicationwith the first proximal end (e.g., a first reservoir fluidicallyconnected to the first proximal end), where the first reservoir includesat least one portion of the first liquid; and v) a second reservoir influid communication with the second proximal end (e.g., a secondreservoir fluidically connected to the second proximal end), where thesecond reservoir comprises at least one portion of the first liquid, andb) a second liquid contained in the droplet formation region, e.g.,where the first liquid and the second liquid are immiscible. Theportions of the first liquid are miscible and combine at theintersection of the first channel and second channel to form the firstliquid.

In some embodiments, a portion of the first liquid in the firstreservoir comprises particles. In certain embodiments, a portion of thefirst liquid in the second reservoir comprises an analyte.

In certain embodiments, the first channel and the droplet formationregion of the device are configured to produce droplets including asingle particle or a single particle of multiple types, e.g., one beadand one cell. In some embodiments, the third width of the deviceincreases from the inlet of the shelf region to the outlet of the shelfregion. In certain embodiments, the device of the system includes acollection reservoir configured to collect droplets formed in thedroplet formation region.

In further embodiments, the device of the system includes a thirdchannel having a third proximal end and a third distal end, where thethird proximal end is in fluid communication with the shelf region andwhere the third distal end is in fluid communication with the stepregion (e.g., the third proximal end is fluidically connected to theshelf region and the third distal end is fluidically connected to thestep region). In further embodiments, the device of the system includesa plurality of first channels, second channels, and droplet formationregions.

The system may also include a controller operatively coupled totransport the portion of the first liquid in the first liquid and theportion of first liquid in the second reservoir to the intersection.

In another related aspect, the invention includes a kit for producingdroplets of a first liquid in a second liquid, the kit including a) adevice for producing droplets, where the device includes i) a firstchannel having a first depth, a first width, a first proximal end, and afirst distal end; ii) a second channel having a second depth, a secondwidth, a second proximal end, and a second distal end, where the secondchannel intersects the first channel between the first proximal andfirst distal ends; iii) a droplet formation region having a shelf regionhaving a third depth and a third width and a step region having a fourthdepth, where the shelf region is configured to allow the first liquid toexpand in at least one dimension and has at least one inlet and at leastone outlet, where the shelf region is disposed between the first distalend and the step region; iv) a first reservoir in fluid communicationwith the first proximal end (e.g., a first reservoir fluidicallyconnected to the first proximal end); and v) a second reservoir in fluidcommunication with the second proximal end (e.g., a second reservoirfluidically connected to the second proximal end); b) a portion of thefirst liquid; and c) a second liquid, e.g., that is immiscible with thefirst liquid. The device is configured to produce droplets of the firstliquid in the second liquid.

In some embodiments, the first liquid contains particles. In certainembodiments, the first channel and the droplet formation region areconfigured to produce droplets including a single particle or a singleparticle of multiple types, e.g., one bead and one cell. In someembodiments, the third width increases from the inlet of the shelfregion to the outlet of the shelf region. In some embodiments, the thirdwidth of the device increases from the inlet of the shelf region to theoutlet of the shelf region.

In further embodiments, the device of the kit includes a collectionreservoir configured to collect droplets formed in the droplet formationregion. In certain embodiments, the device is configured to produce apopulation of droplets that are substantially stationary in thecollection reservoir.

In further embodiments, the device of the kit includes a third channelhaving a third proximal end and a third distal end, where the thirdproximal end is in fluid communication with the shelf region and wherethe third distal end is in fluid communication with the step region(e.g., the third proximal end is fluidically connected to the shelfregion and the third distal end is fluidically connected to the stepregion). In further embodiments, the device of the kit includes aplurality of first channels, second channels, and droplet formationregions.

In another aspect, the invention provides a device for producingdroplets of a first liquid in a second liquid, the device having a) afirst channel having a first depth, a first width, a first proximal end,a first distal end, and a first surface having a first water contactangle; and b) a droplet formation region having a second surface havinga second water contact angle. The droplet formation region may beconfigured to allow the first liquid to expand in at least onedimension. The droplet formation region may have at least one inlet andat least one outlet. The second water contact angle may be greater thanthe first water contact angle. The first channel and droplet formationregion are configured to produce droplets of the first liquid in thesecond liquid.

In some embodiments, the device further includes a second channel havinga second depth, a second width, a second proximal end, and a seconddistal end. The second channel may intersect the first channel betweenthe first proximal and first distal ends.

In certain embodiments, the droplet formation region includes a shelfregion having a third depth and a third width. In particularembodiments, the droplet formation region includes a step region havinga fourth depth.

In further embodiments, the second contact angle is 5° to 100° greaterthan the first contact angle. In yet further embodiments, the secondwater contact angle is at least 100°.

In some embodiments, the device further includes a first reservoir influid communication with the first proximal end (e.g., a first reservoirfluidically connected to the first proximal end). In particularembodiments, the device further includes a second reservoir in fluidcommunication with the second proximal end (e.g., a second reservoirfluidically connected to the second proximal end). In certainembodiments, the device further includes a collection reservoirconfigured to collect droplets formed in the droplet formation region.In further embodiments, the device is configured to produce a populationof droplets that are substantially stationary in the collectionreservoir.

In yet further embodiments, the first liquid includes particles. Instill further embodiments, the first channel and the droplet formationregion are configured to produce droplets including a single particle ora single particle of multiple types, e.g., one bead and one cell.

In another related aspect, the invention provides a system for producingdroplets of a first liquid in a second liquid. In some embodiments, thesystem includes: a) a device for producing droplets, the deviceincluding: i) a first channel having a first depth, a first width, afirst proximal end, a first distal end, and a first surface having afirst water contact angle; ii) a droplet formation region having asecond surface having a second water contact angle; and iii) a firstreservoir in fluid communication with the first proximal end (e.g., afirst reservoir fluidically connected to the first proximal end) andcomprising at least a portion of the first liquid; and b) a secondliquid contained in the droplet formation region. The first liquid andthe second liquid may be immiscible. The droplet formation region may beconfigured to allow the first liquid to expand in at least onedimension. The droplet formation region may have at least one inlet andat least one outlet. The second water contact angle may be greater thanthe first water contact angle. The system may be configured to producedroplets of the first liquid in the second liquid.

In certain embodiments, the first reservoir further includes particles.

In particular embodiments, the device further includes a second channelhaving a second depth, a second width, a second proximal end, and asecond distal end. The second channel may intersect the first channelbetween the first proximal and first distal ends.

In some embodiments, the device further includes a second reservoir influid communication with the second proximal end (e.g., a secondreservoir fluidically connected to the second proximal end) and containsat least one portion of the first liquid. In further embodiment, theportion of the first liquid in the first channel and the portion of thefirst liquid in the second channel combine at the intersection of thefirst channel and second channel to form the first liquid.

In yet further embodiments, the droplet formation region includes ashelf region having a third depth and a third width at or distal to theat least one inlet of the droplet formation region. In still furtherembodiments, the droplet formation region includes a step region havinga fourth depth at or distal to the at least one outlet of the dropletformation region.

In particular embodiments, the second contact angle is 5° to 100°greater than the first contact angle. In certain embodiments, the secondwater contact angle is at least 100°.

In some embodiments, the device further includes a collection reservoirconfigured to collect droplets formed in the droplet formation region.In further embodiments, the device is configured to produce a populationof droplets that are substantially stationary in the collectionreservoir. In yet further embodiments, the system further includes acontroller operatively coupled to transport the portion of the firstliquid in the first reservoir and the portion of first liquid in thesecond reservoir to the intersection.

In another aspect, the invention provides a method of producing amicrofluidic device including a surface modification.

In some embodiments, the method includes: (i) providing a primedmicrofluidic device including a channel in fluid communication with adroplet formation region having a primed surface; and (ii) contactingthe primed surface with a coating agent having affinity for the primedsurface to produce a surface having a water contact angle. The dropletformation region may be configured to allow a liquid exiting the channelto expand in at least one dimension. The contact angle may be greaterthan the water contact angle of the primed surface and greater than thewater contact angle of the channel.

In certain embodiments, the method further includes producing the primedmicrofluidic device by depositing a layer of metal oxide onto anunmodified droplet formation region surface. In particular embodiments,the coating agent is in a coating carrier (e.g., a coating liquid orcoating gas).

In further embodiments, step (ii) includes filling the channel with ablocking liquid that is substantially immiscible with the coatingcarrier (e.g., the coating liquid). Filling the channel with a blockingliquid may substantially prevent ingress of the coating agent into thechannel.

In particular embodiments, step (ii) includes supplying a gas to thechannel, wherein the gas pressure substantially prevents ingress of thecoating agent into the channel.

In some embodiments, the microfluidic device further includes a coatingfeed channel. The coating feed channel may be in fluid communicationwith the droplet formation region. The coating agent may be provided tothe droplet formation region through the coating feed channel.

In another aspect, the invention provides a device for producingdroplets of a first fluid in a second fluid, the device including a) afirst channel having a first depth, a first width, a first proximal end,and a first distal end; b) a second channel having a second depth, asecond width, a second proximal end, and a second distal end, where thesecond channel intersects the first channel between the first proximaland first distal ends; and c) a plurality of droplet formation regions,where the droplet formation region is configured to allow the firstliquid to expand in at least one dimension and has at least one inletand at least one outlet. The first channel and droplet formation regionsare configured to produce droplets of the first liquid in the secondliquid. Devices of this aspect of the invention can include surfaceshaving a surface modification, e.g., alteration to the water contactangle of the surface.

In some embodiments, the first fluid contains particles. In certainembodiments, the first channel and the droplet formation region areconfigured to produce droplets including a single particle or a singleparticle of multiple types, e.g., one bead and one cell.

In some embodiments, at least one of the droplet formation regionsincludes a shelf region having a third depth and a third width.

In some embodiments, at least one of the droplet formation regionsincludes a step region having a fourth depth. In further embodiments, atleast one of the droplet formation regions includes a shelf region thatis disposed between the first distal end and the step region.

In further embodiments, the device includes a collection reservoirconfigured to collect a population of droplets formed in the dropletformation region.

In another embodiment, the device includes a) two first channels, eachhaving a first depth, a first width, a first proximal end, and a firstdistal end; b) two second channels each having a second depth, a secondwidth, a second proximal end, and a second distal end, where each of thesecond distal ends intersects one of the first channels between thefirst proximal and first distal ends and where one of the secondchannels traverses but does not intersect at least one first channel;and c) a plurality of droplet formation regions, where each dropletformation region is configured to allow the first liquid to expand in atleast one dimension and has at least one inlet and at least one outletand each droplet formation region is connected to one of the firstdistal ends. The two first channels and the droplet formation regionsare configured to produce droplets of the first liquid in the secondliquid. Devices of this embodiment of the invention can include surfaceshaving a surface modification, e.g., alteration to the water contactangle of the surface.

In some embodiments, the first fluid contains particles. In certainembodiments, the first channel and the droplet formation region areconfigured to produce droplets including a single particle or a singleparticle of multiple types, e.g., one bead and one cell.

In some embodiments, at least one of the droplet formation regionsincludes a shelf region having a third depth and a third width. In someembodiments, at least one of the droplet formation regions includes astep region having a fourth depth. In further embodiments, at least oneof the droplet formation regions includes a shelf region that isdisposed between the first distal end and the step region.

In further embodiments, the device includes a collection reservoirconfigured to collect a population of droplets formed in the dropletformation region. In some embodiments, the first proximal ends are influid communication with a first reservoir. In some embodiments, thesecond proximal ends are in fluid communication with a second reservoir.

In certain embodiments, the first proximal end of one first channelintersects the other first channel. In certain embodiments, the secondproximal end of one second channel intersects the other second channel.

In yet another embodiment, the device includes a) two first channels,each having a first depth, a first width, a first proximal end, and afirst distal end; b) two second channels each having a second depth, asecond width, a second proximal end, and a second distal end, where thetwo second channels intersect the two first channels between the firstproximal and first distal ends; and c) two droplet formation regions,where the droplet formation regions are configured to allow the firstliquid to expand in at least one dimension and have at least one inletand at least one outlet. The two first channels and the two dropletformation regions are configured to produce droplets of the first liquidin the second liquid. Devices of this embodiment of the invention caninclude surfaces having a surface modification, e.g., alteration to thewater contact angle of the surface.

In some embodiments, the first fluid contains particles.

In some embodiments, the two droplet formation regions include a shelfregion having a third depth and a third width. In further embodiments,the two droplet formation regions include a step region having a fourthdepth.

In further embodiments, the device includes a collection reservoirconfigured to collect a population of droplets formed in the two dropletformation regions. In some embodiments, the first proximal ends are influid communication with a first reservoir. In some embodiments, thesecond proximal ends are in fluid communication with a second reservoir.

In some embodiments, the first proximal end of one first channelintersects the other first channel. In some embodiments, the secondproximal end of one second channel intersects the other second channel.

In a further aspect, the invention provides a method of producingdroplets of a first liquid in a second liquid.

In some embodiments, the method includes a) providing a deviceincluding: i) a first channel having a first proximal end, a firstdistal end, a first depth, and a first width, the first channelcomprising particles and a first liquid; and ii) a droplet formationregion in fluid communication with the first channel; and iii) acollection region configured to collect droplets formed in the dropletformation region and containing the second liquid; and b) allowing thefirst liquid to flow from the first channel to the droplet formationregion to produce droplets of the first liquid and particles in thesecond liquid.

The droplet formation region may be configured to allow the first liquidto expand in at least one dimension. The first liquid may besubstantially immiscible with the second liquid. The device may becapable of forming droplets without externally driving the secondliquid.

In some embodiments, the droplets are substantially stationary in thecollection region.

In further embodiments, the first depth decreases in theproximal-to-distal direction (e.g., in the flow direction, e.g., towardsthe droplet formation region) in at least a portion of the firstchannel. In yet further embodiments, the first depth increases in theproximal-to-distal direction (e.g., in the flow direction, e.g., towardsthe droplet formation region) in at least a portion of the firstchannel. In still further embodiments, the first channel furthercomprises a groove.

In certain embodiments, the device further includes a first reservoir influid communication with the first proximal end (e.g., a first reservoirfluidically connected to the first proximal end). In particularembodiments, the first reservoir further contains the particles.

In some embodiments, step b) produces droplets having a single particleor a single particle of multiple types, e.g., one bead and one cell.

In other embodiments, the particles have about the same density as thefirst liquid. In yet other embodiments, the density of the first liquidis lower than the density of the second liquid. In still otherembodiments, the density of the first liquid is higher than the densityof the second liquid.

In particular embodiments, the first liquid is aqueous or miscible withwater.

In some embodiments, the device further includes a second channel havinga second proximal end, a second distal end, a second depth, and a secondwidth. The second channel may intersect the first channel between thefirst proximal end and the first distal end. The second channel mayinclude a third liquid. The third liquid may combine with the firstliquid at the intersection, and the droplets may further contain thethird liquid.

In further embodiments, the second depth decreases in theproximal-to-distal direction (e.g., in the flow direction, e.g., towardsthe droplet formation region) in at least a portion of the secondchannel. In yet further embodiments, the second depth increases in theproximal-to-distal direction (e.g., in the flow direction, e.g., towardsthe droplet formation region) in at least a portion of the secondchannel.

In still further embodiments, the third liquid is aqueous or misciblewith water. In some embodiments, the density of the third liquid islower than the density of the second liquid. In certain embodiments, thedensity of the third liquid is higher than the density of the secondliquid.

In particular embodiments, the device further includes a secondreservoir in fluid communication with the second proximal end (e.g., asecond reservoir fluidically connected to the second proximal end).

In some embodiments, the second channel further includes a groove. Incertain embodiments, the droplet formation region comprises a shelfregion having a third depth and a third width. The shelf region has atleast one inlet and at least one outlet.

In certain embodiments, the third width increases from the inlet of theshelf region to the outlet of the shelf region.

In particular embodiments, the droplet formation region includes a stepregion having a fourth depth.

In some embodiments, the droplet formation region further includes ashelf region that is disposed between the first distal end and the stepregion.

In certain embodiments, the device further includes a third channelhaving a third proximal end and a third distal end. The third proximalend may be in fluid communication with the shelf region. The thirddistal end may be in fluid communication with the step region.

In further embodiments, the droplet formation region includes aplurality of inlets in fluid communication with the first proximal endand a plurality of outlets (e.g., fluidically connected to the firstproximal end and in fluid communication with a plurality of outlets). Inyet further embodiments, the number of inlets and the number of outletsis the same.

In another aspect, the invention features a method of producing ananalyte detection droplet. The method includes providing a device havinga plurality of particles in a liquid carrier, wherein the particlesinclude an analyte detection moiety. The device also includes a sampleliquid having an analyte, a particle channel, a sample channel thatintersects with the particle channel at an intersection, a dropletformation region distal to the particle channel and the sample channel,and a droplet collection region. The droplet formation region isconfigured to allow the liquid carrier to expand in at least onedimension and can include a step. Particles in the liquid carrier flowproximal-to-distal (e.g., in the flow direction, e.g., towards thedroplet formation region) through the particle channel, and the sampleliquid is allowed to flow proximal-to-distal (e.g., in the flowdirection, e.g., towards the droplet formation region) through thesample channel. The sample liquid combines with the particles in theliquid carrier to form an analyte detection liquid at the intersection,and the analyte detection liquid meets a partitioning liquid at thedroplet formation region under droplet forming conditions to form aplurality of analyte detection droplets.

The plurality of analyte detection droplets includes one or more of theparticles in the analyte detection liquid (e.g., one or more of theplurality of analyte detection droplets includes one or more particles).

In some embodiments, the particle channel is one of a plurality ofparticle channels and the sample channel is one of a plurality of samplechannels. The device can further include a particle reservoir connectedproximally to the plurality of particle channels and a sample reservoirconnected proximally to the plurality of sample channels.

In some embodiments, the sample liquid and the liquid carrier aremiscible. In some embodiments, the sample liquid and the liquid carrierare aqueous liquids and the partitioning liquid is immiscible with thesample liquid and the liquid carrier. The analyte can be a bioanalyte,for example, a nucleic acid, an intracellular protein, a glycan, or asurface protein. The analyte detection moiety can include a nucleic acidor an antigen-binding protein. The sample can include a cell, or acomponent or product thereof. In some embodiments, the plurality ofanalyte detection droplets accumulates as a population (e.g., asubstantially stationary population) in the droplet collection region.

In another aspect, the invention provides a method of producing abioanalyte detection droplet by providing a device having a plurality ofparticles in an aqueous carrier, a particle channel, a droplet formationregion, and a droplet collection region. The particles can include abioanalyte detection moiety, the droplet formation region is configuredto allow the aqueous carrier to expand in at least one dimension, theparticle channel is proximal to the droplet formation region, and thedroplet formation region is proximal to the droplet collection region.The method further includes allowing the particles in the aqueouscarrier to flow proximal-to-distal (e.g., towards the droplet formationregion) through the particle channel and droplet formation region. Theaqueous carrier meets a partitioning liquid at the droplet formationregion under droplet forming conditions, thereby forming a plurality ofbioanalyte detection droplets. The plurality of bioanalyte detectiondroplets includes one or more of the particles in the aqueous carrier(e.g., one or more of the plurality of bioanalyte detection dropletsincludes one or more particles), and the plurality of bioanalytedetection droplets accumulate in the droplet collection region. In someembodiments, the device further includes a sample channel thatintersects with the particle channel proximal to the droplet formationregion at an intersection. The aqueous sample including a bioanalyteflows proximal-to-distal (e.g., towards the droplet formation region)through the sample channel and combines with particles in the aqueouscarrier at the intersection. The plurality of bioanalyte detectiondroplets includes the aqueous sample and one or more particles in theaqueous carrier. For example, one or more of each of the plurality ofbioanalyte detection droplets includes one or more particles. Devices ofthis aspect of the invention can include any one or more features of anyof the devices from any of the preceding aspects.

In some embodiments, the droplet formation region includes a step. Insome embodiments, the particle channel is one of a plurality of particlechannels and the sample channel is one of a plurality of samplechannels. In some embodiments, the device further includes a particlereservoir connected proximally to the plurality of particle channels anda sample reservoir connected proximally to the plurality of samplechannels.

The bioanalyte detection moiety can include a nucleic acid and/or abarcode. In some embodiments, the bioanalyte is selected from the groupconsisting of a surface-expressed protein, an intracellular protein, aglycan, and a nucleic acid.

In some embodiments, the aqueous sample includes a cell, or a componentor product thereof. In some embodiments, the aqueous carrier includesone or more enzymes and/or lysis agents.

The method can further include, after the bioanalyte detection dropletsare formed, incubating the droplets under conditions sufficient to allowthe bioanalyte detection moiety to label the bioanalyte. In someembodiments, the bioanalyte is a nucleic acid, and after labeling thebioanalyte, incubating the reaction droplets under conditions sufficientto amplify the barcoded nucleic acids. In some embodiments, the aqueouscarrier includes one or more enzymes, such as reverse transcriptase.

In some embodiments, the particle channel is one of a plurality ofparticle channels and the sample channel is one of a plurality of samplechannels, and the device further includes a particle reservoir connectedproximally to the plurality of particle channels and a sample reservoirconnected proximally to the plurality of sample channels.

In yet another aspect, the invention features a method of barcoding apopulation of cells by providing a device having a plurality ofparticles in an aqueous carrier, an aqueous sample having a populationof cells, a particle channel, a sample channel, a droplet formationregion (e.g., a droplet formation region including a step), and adroplet collection region. The particles can include a nucleic acidprimer sequence and a barcode, and the droplet formation region isconfigured to allow the aqueous carrier to expand in at least onedimension. The particle channel intersects the sample channel at anintersection proximal to the droplet formation region, and the dropletformation region is proximal to the droplet collection region. Theparticles in the aqueous carrier flow proximal-to-distal (e.g., towardsthe droplet formation region) through the particle channel, and theaqueous sample is allowed to flow proximal-to-distal (e.g., towards thedroplet formation region) through the sample channel. The aqueous samplecombines with the particles in the aqueous carrier to form a reactionliquid at the intersection, and the reaction liquid meets a partitioningliquid at the droplet formation region under droplet forming conditionsto form a plurality of reaction droplets. The plurality of reactiondroplets includes one or more of the particles in the reaction liquid(e.g., one or more of the plurality of reaction droplets includes one ormore particles). The plurality of reaction droplets accumulates in thedroplet collection region, and the reaction droplets are incubated underconditions sufficient to allow for barcoding nucleic acids in thepopulation of cells.

In some embodiments, the particle channel is one of a plurality ofparticle channels and the sample channel is one of a plurality of samplechannels, and the device further includes a particle reservoir connectedproximally to the plurality of particle channels and a sample reservoirconnected proximally to the plurality of sample channels. Devices ofthis aspect of the invention can include any one or more features of anyof the devices from any of the preceding aspects.

In some embodiments, the aqueous carrier includes a lysis reagentconfigured to lyse the cells before or during the incubation of thereaction droplets. The aqueous carrier or the aqueous sample can includeone or more enzymes, such as reverse transcriptase.

In another embodiment, the invention provides a method of single-cellnucleic acid sequencing. The method includes providing a device having aplurality of particles in an aqueous carrier, an aqueous sample having apopulation of cells, a particle channel, a sample channel, a dropletformation region (e.g., a droplet formation region including a step),and a droplet collection region. The particles include a nucleic acidprimer sequence and a barcode, and the droplet formation region isconfigured to allow the aqueous carrier to expand in at least onedimension. The particle channel intersects the sample channel at anintersection proximal to the droplet formation region, and the dropletformation region is proximal to the droplet collection region. Theparticles in the aqueous carrier flow proximal-to-distal (e.g., towardsthe droplet formation region) through the particle channel, and theaqueous sample is allowed to flow proximal-to-distal (e.g., towards thedroplet formation region) through the sample channel. The aqueous samplecombines with the particles in the liquid carrier to form a reactionliquid at the intersection, and the reaction liquid meets a partitioningliquid at the droplet formation region under droplet forming conditionsto form a plurality of reaction droplets. The plurality of reactiondroplets includes one or more of the particles and a single cell orlysate thereof (e.g., one or more of the plurality of reaction dropletsincludes one or more particles and a single cell or lysate thereof). Insome embodiments, one or more (e.g., at least 1%, 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%) of the reactiondroplets includes a single particle and a single cell. The plurality ofdroplets accumulates in the droplet collection region. The reactiondroplets are incubated under conditions sufficient to generate barcodednucleic acids, and the barcoded nucleic acid transcripts are sequencedto obtain nucleic acid sequences associated with single cells. Devicesof this aspect of the invention can include any one or more features ofany of the devices from any of the preceding aspects.

In some embodiments, the aqueous carrier includes a lysis reagentconfigured to lyse the cells before or during incubation of the reactiondroplets. The aqueous carrier or the aqueous sample can include one ormore enzymes, such as reverse transcriptase. In some embodiments, themethod further includes compiling the nucleic acid sequences associatedwith single cells into a genome library.

In yet another aspect, the invention provides a device for producingdroplets of a first fluid in a second fluid.

In one embodiment, the device includes a) a first channel having a firstdepth, a first width, a first proximal end, and a first distal end; andb) a droplet formation region in fluid communication with, e.g.,fluidically connected to, the first distal end, wherein the dropletformation region comprises a shelf region having a second depth and asecond width and a step region having a third depth, wherein the secondwidth is greater than the first width and increases from the firstdistal end towards the step region, the third depth is greater than thefirst depth, and the shelf region is disposed between the first distalend and the step region. The first channel and droplet formation regionare configured to produce droplets of the first fluid in the secondfluid, e.g., as a result of the first fluid flowing from the firstdistal end to the step region.

In another embodiment, the device includes a) a first channel having afirst depth, a first width, a first proximal end, and a first distalend; b) a second channel having a fourth depth, a fourth width, a fourthproximal end, and a fourth distal end, wherein the second channelintersects the first channel between the first proximal and first distalends; and c) a droplet formation region in fluid communication with,e.g., fluidically connected to, the first distal end, wherein thedroplet formation region comprises a shelf region having a second depthand a second width and a step region having a third depth, wherein thesecond width is greater than the first width, the third depth is greaterthan the first depth, and the shelf region is disposed between the firstdistal end and the step region. The first channel and droplet formationregion are configured to produce droplets of the first fluid in thesecond fluid, e.g., as a result of the first fluid flowing from thefirst distal end to the step region.

In a further embodiment, the device includes a) a first channel having afirst depth, a first width, a first proximal end, and a first distalend; b) a droplet formation region fluidically connected to the firstdistal end, wherein the droplet formation region comprises a shelfregion having a second depth and a second width and a step region havinga third depth, wherein the second width is greater than the first width,the third depth is greater than the first depth, and the shelf region isdisposed between the first distal end and the step region; and c) athird channel having an outlet into the shelf region between the firstdistal end and the step region. The first channel and droplet formationregion are configured to produce droplets of the first fluid in thesecond fluid, e.g., as a result of the first fluid flowing from thefirst distal end to the step region.

In various embodiments of the devices of the invention, the first fluidincludes particles, such as gel beads. In these embodiments, the firstchannel and the droplet formation region may be configured to producedroplets including a single particle or a single particle of multipletypes, e.g., one bead and one cell. In other embodiments, the firstchannel further includes a groove. In other embodiments, the devicefurther includes a first reservoir to which the first proximal end isfluidically connected; a second reservoir to which the shelf region isin fluid communication with, e.g., fluidically connected to; and/or athird reservoir to which the step region is in fluid communication with,e.g., fluidically connected to. In some embodiments, the first depth andthe second depth are the same. In further embodiments, the first depthis greater than the second depth.

In embodiments of certain devices, the devices further include a secondchannel having a fourth depth, a fourth width, a fourth proximal end,and a fourth distal end, wherein the second channel intersects the firstchannel between the first proximal and first distal ends. In theseembodiments, the device may also include a fourth reservoir to which thefourth proximal end is fluidically connected.

In embodiments of certain devices, the devices further include a thirdchannel having an outlet to the shelf region between the first distalend and the step region.

Devices of the invention may also include a controller operativelycoupled to the first channel to transport the first fluid out of thefirst distal end.

In certain embodiments, the first channel further includes a pluralityof distal ends each fluidically connected to the droplet formationregion.

In further embodiments, the second depth is substantially constant. Inother embodiments, the second depth increases from the first distal endtowards the step region. In further embodiments, the third depth issubstantially constant. In other embodiments, the third depth increasesaway from the shelf region.

In devices of the invention, the step region may increase in depthupward from the shelf region, downward from the shelf region, or both.

In further embodiments, the second width is greater than the first widthand increases from the first distal end towards the step region. Thesecond width may increase linearly or non-linearly.

In other embodiments, the shelf region or step region further includes asurface coating.

In yet other embodiments, the first channel and the shelf region arecombined to form a merged channel that increases in width from the firstproximal end towards the step region.

In another aspect, the invention provides a method of forming a dropletof a first fluid in a second fluid. In one embodiment, the methodincludes transporting a first fluid, e.g., one including particles,through a channel into a second fluid that is stationary underconditions that droplets form.

In other embodiments, the method includes providing a device of theinvention and flowing the first fluid through the first channel to thestep region, thereby forming the droplet of the first fluid.

In further embodiments, the method includes transporting the first fluidthrough a channel having a change in width along its length so that adroplet forms as the first fluid passes along the channel into thesecond fluid, wherein the droplet comprises a particle.

In embodiments of the methods of the invention, the channel is a firstchannel having a first depth, a first width, a first proximal end, and afirst distal end and disposed in a device; and the device furtherincludes a droplet formation region fluidically connected to the firstdistal end, wherein the droplet formation region includes a shelf regionhaving a second depth and a second width and a step region having athird depth, wherein the second width is greater than the first width,the third depth is greater than the first depth, and the shelf region isdisposed between the first distal end and the step region.

In certain embodiments, the first fluid and second fluid are immiscible.The first fluid may be aqueous, and/or the second fluid includes an oil.The first fluid may include particles, e.g., gel beads. In certainembodiments, the first channel and the droplet formation region areconfigured to produce droplets including a single particle or a singleparticle of multiple types, e.g., one bead and one cell. The diameter ofthe gel beads may be substantially similar to the dimensions of thefirst channel. In other embodiments, the diameter of the gel beads issubstantially larger than the dimensions of the first channel.

It will be understood that the devices, systems, kits, and methodsdescribed herein may, in addition to features specified, include anyfeature described herein that is not inconsistent with the structure ofthe underlying device, system, kit, or method. Thus, devices may includemultiple drop formation regions, either in communication with each otheror not in fluid communication with either other, differential surfacefeatures, or additional elements or steps as described herein.

Definitions

Where values are described as ranges, it will be understood that suchdisclosure includes the disclosure of all possible sub-ranges withinsuch ranges, as well as specific numerical values that fall within suchranges irrespective of whether a specific numerical value or specificsub-range is expressly stated.

The terms “adaptor(s)”, “adapter(s)” and “tag(s)” may be usedsynonymously. An adaptor or tag can be coupled to a polynucleotidesequence to be “tagged” by any approach including ligation,hybridization, or other approaches.

The term “barcode,” as used herein, generally refers to a label, oridentifier, that conveys or is capable of conveying information about ananalyte. A barcode can be part of an analyte. A barcode can be a tagattached to an analyte (e.g., nucleic acid molecule) or a combination ofthe tag in addition to an endogenous characteristic of the analyte(e.g., size of the analyte or end sequence(s)). A barcode may be unique.Barcodes can have a variety of different formats. For example, barcodescan include:

polynucleotide barcodes; random nucleic acid and/or amino acidsequences; and synthetic nucleic acid and/or amino acid sequences. Abarcode can be attached to an analyte in a reversible or irreversiblemanner. A barcode can be added to, for example, a fragment of adeoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sample before,during, and/or after sequencing of the sample. Barcodes can allow foridentification and/or quantification of individual sequencing-reads inreal time.

The term “bead,” as used herein, generally refers to a generallyspherical or ellipsoid particle that is not a biological particle. Thebead may be a solid or semi-solid particle. The bead may be a gel bead(e.g., a hydrogel bead). The bead may be formed of a polymeric material.The bead may be magnetic or non-magnetic.

The term “biological particle,” as used herein, generally refers to adiscrete biological system derived from a biological sample, such as acell or a particulate component of a cell, such as an organelle,exosome, or vesicle. The biological particle may be a rare cell from apopulation of cells. The biological particle may be any type of cell,including without limitation prokaryotic cells, eukaryotic cells,bacterial, fungal, plant, mammalian, or other animal cell types,mycoplasmas, normal tissue cells, tumor cells, or any other cell type,whether derived from single cell or multicellular organisms. Thebiological particle may be obtained from a tissue of a subject. Thebiological particle may be a hardened cell. Such hardened cell may ormay not include a cell wall or cell membrane. The biological particlemay include one or more constituents of a cell, but may not includeother constituents of the cell. An example of such constituents is anucleus or an organelle. A cell may be a live cell. The live cell may becapable of being cultured, for example, being cultured when enclosed ina gel or polymer matrix, or cultured when comprising a gel or polymermatrix. Alternatively, the biological particle may be a virus.

The term “fluidically connected”, as used herein, refers to a directconnection between at least two device elements, e.g., a channel,reservoir, etc., that allows for fluid to move between such deviceelements without passing through an intervening element.

The term “genome,” as used herein, generally refers to genomicinformation from a subject, which may be, for example, at least aportion or an entirety of a subject's hereditary information. A genomecan be encoded either in DNA or in RNA. A genome can comprise codingregions that code for proteins as well as non-coding regions. A genomecan include the sequence of all chromosomes together in an organism. Forexample, the human genome has a total of 46 chromosomes. The sequence ofall of these together may constitute a human genome.

The term “in fluid communication with”, as used herein, refers to aconnection between at least two device elements, e.g., a channel,reservoir, etc., that allows for fluid to move between such deviceelements with or without passing through one or more intervening deviceelements.

The term “macromolecular constituent,” as used herein, generally refersto a macromolecule contained within a biological particle. Themacromolecular constituent may comprise a nucleic acid. Themacromolecular constituent may comprise deoxyribonucleic acid (DNA). Themacromolecular constituent may comprise ribonucleic acid (RNA). Themacromolecular constituent may comprise a protein. The macromolecularconstituent may comprise a peptide. The macromolecular constituent maycomprise a polypeptide.

The term “molecular tag,” as used herein, generally refers to a moleculecapable of binding to a macromolecular constituent. The molecular tagmay bind to the macromolecular constituent with high affinity. Themolecular tag may bind to the macromolecular constituent with highspecificity. The molecular tag may comprise a nucleotide sequence. Themolecular tag may comprise an oligonucleotide or polypeptide sequence.The molecular tag may comprise a DNA aptamer. The molecular tag may beor comprise a primer. The molecular tag may be or comprise a protein.The molecular tag may comprise a polypeptide. The molecular tag may be abarcode.

The term “oil,” as used herein, generally refers to a liquid that is notmiscible with water. An oil may have a density higher or lower thanwater and/or a viscosity higher or lower than water.

The term “sample,” as used herein, generally refers to a biologicalsample of a subject. The biological sample may be a nucleic acid sampleor protein sample. The biological sample may be derived from anothersample. The sample may be a tissue sample, such as a biopsy, corebiopsy, needle aspirate, or fine needle aspirate. The sample may be aliquid sample, such as a blood sample, urine sample, or saliva sample.The sample may be a skin sample. The sample may be a cheek swap. Thesample may be a plasma or serum sample. The sample may include abiological particle, e.g., a cell or virus, or a population thereof, orit may alternatively be free of biological particles. A cell-free samplemay include polynucleotides. Polynucleotides may be isolated from abodily sample that may be selected from the group consisting of blood,plasma, serum, urine, saliva, mucosal excretions, sputum, stool andtears.

The term “sequencing,” as used herein, generally refers to methods andtechnologies for determining the sequence of nucleotide bases in one ormore polynucleotides. The polynucleotides can be, for example,deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), includingvariants or derivatives thereof (e.g., single stranded DNA). Sequencingcan be performed by various systems currently available, such as,without limitation, a sequencing system by Illumina, PacificBiosciences, Oxford Nanopore, or Life Technologies (Ion Torrent). As analternative, sequencing may be performed using nucleic acidamplification, polymerase chain reaction (PCR) (e.g., digital PCR,quantitative PCR, or real time PCR) or isothermal amplification. Suchdevices may provide a plurality of raw genetic data corresponding to thegenetic information of a subject (e.g., human), as generated by thedevice from a sample provided by the subject. In some situations,systems and methods provided herein may be used with proteomicinformation.

The term “subject,” as used herein, generally refers to an animal, suchas a mammal (e.g., human) or avian (e.g., bird), or other organism, suchas a plant. The subject can be a vertebrate, a mammal, a mouse, aprimate, a simian or a human. Animals may include, but are not limitedto, farm animals, sport animals, and pets. A subject can be a healthy orasymptomatic individual, an individual that has or is suspected ofhaving a disease (e.g., cancer) or a pre-disposition to the disease, oran individual that is in need of therapy or suspected of needingtherapy. A subject can be a patient.

The term “substantially stationary”, as used herein with respect todroplet formation, generally refers to a state when motion of formeddroplets in the continuous phase is passive, e.g., resulting from thedifference in density between the dispersed phase and the continuousphase.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an example of a microfluidic device for the introduction ofparticles, e.g., beads, into discrete droplets.

FIG. 2 shows an example of a microfluidic device for increased dropletformation throughput.

FIG. 3 shows another example of a microfluidic device for increaseddroplet formation throughput.

FIG. 4 shows another example of a microfluidic device for theintroduction of particles, e.g., beads, into discrete droplets.

FIGS. 5A-5B show cross-section (FIG. 5A) and perspective (FIG. 5B) viewsan embodiment according to the invention of a microfluidic device with ageometric feature for droplet formation.

FIGS. 6A-6B show a cross-section view and a top view, respectively, ofanother example of a microfluidic device with a geometric feature fordroplet formation.

FIGS. 7A-7B show a cross-section view and a top view, respectively, ofanother example of a microfluidic device with a geometric feature fordroplet formation.

FIGS. 8A-8B show a cross-section view and a top view, respectively, ofanother example of a microfluidic device with a geometric feature fordroplet formation.

FIGS. 9A-9B are views of another device of the invention. FIG. 9A is topview of a device of the invention with reservoirs. FIG. 9B is amicrograph of a first channel intersected by a second channel adjacent adroplet formation region.

FIGS. 10A-10E are views of droplet formation regions including shelfregions.

FIGS. 11A-11D are views of droplet formation regions including shelfregions including additional channels to deliver continuous phase.

FIG. 12 is another device according to the invention having a pair ofintersecting channels that lead to a droplet formation region andcollection reservoir.

FIGS. 13A-13B are views of a device of the invention. FIG. 13A is anoverview of a device with four droplet formation regions. FIG. 13B is azoomed in view of an exemplary droplet formation region within thedotted line box in FIG. 13A.

FIGS. 14A-14B are views of devices according to the invention. FIG. 14Ashows a device with three reservoirs employed in droplet formation. FIG.14B is a device of the invention with four reservoirs employed in thedroplet formation.

FIG. 15 is a view of a device according to the invention with fourreservoirs.

FIGS. 16A-16B are views of an embodiment according to the invention.FIG. 16A is a top view of a device having two liquid channels that meetadjacent to a droplet formation region. FIG. 16B is a zoomed in view ofthe droplet formation region showing the individual droplet formationsregions.

FIGS. 17A-17B are schematic representations of a method according to theinvention for applying a differential coating to a surface of a deviceof the invention. FIG. 17A is an overview of the method, and FIG. 17B isa micrograph showing the use of a blocking fluid to protect a channelfrom a coating agent.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides devices, kits, and systems for forming dropletsand methods of their use. The devices may be used to form droplets of asize suitable for utilization as microscale chemical reactors, e.g., forgenetic sequencing. In general, droplets are formed in a device byflowing a first liquid through a channel and into a droplet formationregion including a second liquid, i.e., the continuous phase, which mayor may not be externally driven. Thus, droplets can be formed withoutthe need for externally driving the second liquid.

In the present invention, the size of the generated droplets issignificantly less sensitive to changes in liquid properties. Forexample, the size of the generated droplets is less sensitive to thedispersed phase flow rate. Adding multiple formation regions is alsosignificantly easier from a layout and manufacturing standpoint. Theaddition of further formation regions allows for formation of dropletseven in the event that one droplet formation region becomes blocked.Droplet formation can be controlled by adjusting one or more geometricfeatures of fluidic channel architecture, such as a width, height,and/or expansion angle of one or more fluidic channels. For example,droplet size and speed of droplet formation may be controlled. In someinstances, the number of regions of formation at a driven pressure canbe increased to increase the throughput of droplet formation.

Devices

A device of the invention includes a first channel having a depth, awidth, a proximal end, and a distal end. The proximal end is or isconfigured to be in fluid communication with a source of liquid, e.g., areservoir integral to the device or coupled to the device, e.g., bytubing. The distal end is in fluid communication with, e.g., fluidicallyconnected to, a droplet formation region. A droplet formation regionallows liquid from the first channel to expand in at least onedimension, leading to droplet formation under appropriate conditions asdescribed herein. A droplet formation region can be of any suitablegeometry. In one embodiment, the droplet formation region includes ashelf region that allows liquid to expand substantially in onedimension, e.g., perpendicular to the direction of flow. The width ofthe shelf region is greater than the width of the first channel at itsdistal end. In certain embodiments, the first channel is a channeldistinct from a shelf region, e.g., the shelf region widens or widens ata steeper slope or curvature than the distal end of the first channel.In other embodiments, the first channel and shelf region are merged intoa continuous flow path, e.g., one that widens linearly or non-linearlyfrom its proximal end to its distal end; in these embodiments, thedistal end of the first channel can be considered to be an arbitrarypoint along the merged first channel and shelf region. In anotherembodiment, the droplet formation region includes a step region, whichprovides a spatial displacement and allows the liquid to expand in morethan one dimension. The spatial displacement may be upward or downwardor both relative to the channel. The choice of direction may be madebased on the relative density of the dispersed and continuous phases,with an upward step employed when the dispersed phase is less dense thanthe continuous phase and a downward step employed when the dispersedphase is denser than the continuous phase. Droplet formation regions mayalso include combinations of a shelf and a step region, e.g., with theshelf region disposed between the channel and the step region.

Without wishing to be bound by theory, droplets of a first liquid can beformed in a second liquid in the devices of the invention by flow of thefirst liquid from the distal end into the droplet formation region. Inembodiments with a shelf region and a step region, the stream of firstliquid expands laterally into a disk-like shape in the shelf region. Asthe stream of first liquid continues to flow across the shelf region,the stream passes into the step region wherein the droplet assumes amore spherical shape and eventually detaches from the liquid stream. Asthe droplet is forming, passive flow of the continuous phase around thenascent droplet occurs, e.g., into the shelf region, where it reformsthe continuous phase as the droplet separates from its liquid stream.Droplet formation by this mechanism can occur without externally drivingthe continuous phase, unlike in other systems. It will be understoodthat the continuous phase may be externally driven during dropletformation, e.g., by gently stirring or vibration but such motion is notnecessary for droplet formation.

Passive flow of the continuous phase may occur simply around the nascentdroplet. The droplet formation region may also include one or morechannels that allow for flow of the continuous phase to a locationbetween the distal end of the first channel and the bulk of the nascentdroplet. These channels allow for the continuous phase to flow behind anascent droplet, which modifies (e.g., increase or decreases) the rateof droplet formation. Such channels may be fluidically connected to areservoir of the droplet formation region or to different reservoirs ofthe continuous phase. Although externally driving the continuous phaseis not necessary, external driving may be employed, e.g., to pumpcontinuous phase into the droplet formation region via additionalchannels. Such additional channels may be to one or both lateral sidesof the nascent droplet or above or below the plane of the nascentdroplet.

In general, the components of a device, e.g., channels, may have certaingeometric features that at least partly determine the sizes of thedroplets. For example, any of the channels described herein have adepth, a height, h₀, and width, w. The droplet formation region may havean expansion angle, α. Droplet size may decrease with increasingexpansion angle. The resulting droplet radius, R_(d), may be predictedby the following equation for the aforementioned geometric parameters ofh₀, w, and α:

$R_{d} \approx {0.44\left( {1 + {2.2\sqrt{\tan \mspace{14mu} \alpha}\frac{w}{h_{0}}}} \right)\frac{h_{0}}{\sqrt{\tan \mspace{14mu} \alpha}}}$

As a non-limiting example, for a channel with w=21 μm, h =21 μm, andα=3°, the predicted droplet size is 121 μm. In another example, for achannel with w=25 μm, h =25 μm, and α=5°, the predicted droplet size is123 μm. In yet another example, for a channel with w=28 μm, h =28 μm,and α=7°, the predicted droplet size is 124 μm. In some instances, theexpansion angle may be between a range of from about 0.5° to about 4°,from about 0.1° to about 10°, or from about 0° to about 90°. Forexample, the expansion angle can be at least about 0.01°, 0.1°, 0.2°,0.3°, 0.4°, 0.5°, 0.6°, 0.7°, 0.8°, 0.9°, 1°, 2°, 3°, 4°, 5°, 6°, 7°,8°, 9°, 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45°, 50°, 55°, 60°, 65°, 70°,75°, 80°, 85°, or higher. In some instances, the expansion angle can beat most about 89°, 88°, 87°, 86°, 85°, 84°, 83°, 82°, 81°, 80°, 75°,70°, 65°, 60°, 55°, 50°, 45°, 40°, 35°, 30°, 25°, 20°, 15°, 10°, 9°, 8°,7°, 6°, 5°, 4°, 3°, 2°, 1°, 0.1°, 0.01°, or less.

The depth and width of the first channel may be the same, or one may belarger than the other, e.g., the width is larger than the depth, orfirst depth is larger than the width. In some embodiments, the depthand/or width is between about 0.1 μm and 1000 μm. In some embodiments,the depth and/or width of the first channel is from 1 to 750 μm, 1 to500 μm, 1 to 250 μm, 1 to 100 μm, 1 to 50 μm, or 3 to 40 μm. In somecases, when the width and length differ, the ratio of the width to depthis, e.g., from 0.1 to 10, e.g., 0.5 to 2 or greater than 3, such as 3 to10, 3 to 7, or 3 to 5. The width and depths of the first channel may ormay not be constant over its length. In particular, the width mayincrease or decrease adjacent the distal end. In general, channels maybe of any suitable cross section, such as a rectangular, triangular, orcircular, or a combination thereof. In particular embodiments, a channelmay include a groove along the bottom surface. The width or depth of thechannel may also increase or decrease, e.g., in discrete portions, toalter the rate of flow of liquid or particles or the alignment ofparticles.

Devices of the invention may also include additional channels thatintersect the first channel between its proximal and distal ends, e.g.,one or more second channels having a second depth, a second width, asecond proximal end, and a second distal end. Each of the first proximalend and second proximal ends are or are configured to be in fluidcommunication with, e.g., fluidically connected to, a source of liquid,e.g., a reservoir integral to the device or coupled to the device, e.g.,by tubing. The inclusion of one or more intersection channels allows forsplitting liquid from the first channel or introduction of liquids intothe first channel, e.g., that combine with the liquid in the firstchannel or do not combine with the liquid in the first channel, e.g., toform a sheath flow. Channels can intersect the first channel at anysuitable angle, e.g., between 5° and 135° relative to the centerline ofthe first channel, such as between 75° and 115° or 85° and 95°.Additional channels may similarly be present to allow introduction offurther liquids or additional flows of the same liquid. Multiplechannels can intersect the first channel on the same side or differentsides of the first channel. When multiple channels intersect ondifferent sides, the channels may intersect along the length of thefirst channel to allow liquid introduction at the same point.Alternatively, channels may intersect at different points along thelength of the first channel. In some instances, a channel configured todirect a liquid comprising a plurality of particles may comprise one ormore grooves in one or more surface of the channel to direct theplurality of particles towards the droplet formation fluidic connection.For example, such guidance may increase single occupancy rates of thegenerated droplets. These additional channels may have any of thestructural features discussed above for the first channel.

Devices may include multiple first channels, e.g., to increase the rateof droplet formation. In general, throughput may significantly increaseby increasing the number of droplet formation regions of a device. Forexample, a device having five droplet formation regions may generatefive times as many droplets than a device having one droplet formationregion, provided that the liquid flow rate is substantially the same. Adevice may have as many droplet formation regions as is practical andallowed for the size of the source of liquid, e.g., reservoir. Forexample, the device may have at least about 2, 3, 4, 5, 6, 7, 8, 9, 10,20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450,500, 600, 700, 800, 900, 1000, 1500, 2000 or more droplet formationregions. Inclusion of multiple droplet formation regions may require theinclusion of channels that traverse but do not intersect, e.g., the flowpath is in a different plane. Multiple first channel may be in fluidcommunication with, e.g., fluidically connected to, a separate sourcereservoir and/or a separate droplet formation region. In otherembodiments, two or more first channels are in fluid communication with,e.g., fluidically connected to, the same fluid source, e.g., where themultiple first channels branch from a single, upstream channel. Thedroplet formation region may include a plurality of inlets in fluidcommunication with the first proximal end and a plurality of outlets(e.g., plurality of outlets in fluid communication with a collectionregion) (e.g., fluidically connected to the first proximal end and influid communication with a plurality of outlets). The number of inletsand the number of outlets in the droplet formation region may be thesame (e.g., there may be 3-10 inlets and/or 3-10 outlets). Alternativelyor in addition, the throughput of droplet formation can be increased byincreasing the flow rate of the first liquid. In some cases, thethroughput of droplet formation can be increased by having a pluralityof single droplet forming devices, e.g., devices with a first channeland a droplet formation region, in a single device, e.g., paralleldroplet formation.

The width of a shelf region may be from 0.1 μm to 1000 μm. In particularembodiments, the width of the shelf is from 1 to 750 μm, 10 to 500 μm,10 to 250 μm, or 10 to 150 μm. The width of the shelf region may beconstant along its length, e.g., forming a rectangular shape.Alternatively, the width of the shelf region may increase along itslength away from the distal end of the first channel. This increase maybe linear, nonlinear, or a combination thereof. In certain embodiments,the shelf widens 5% to 10,000%, e.g., at least 300%, (e.g., 10% to 500%,100% to 750%, 300% to 1000%, or 500% to 1000%) relative to the width ofthe distal end of the first channel. The depth of the shelf can be thesame as or different from the first channel. For example, the bottom ofthe first channel at its distal end and the bottom of the shelf regionmay be coplanar. Alternatively, a step or ramp may be present where thedistal end meets the shelf region. The depth of the distal end may alsobe greater than the shelf region, such that the first channel forms anotch in the shelf region. The depth of the shelf may be from 0.1 to1000 μm, e.g., 1 to 750 μm, 1 to 500 μm, 1 to 250 μm, 1 to 100 μm, 1 to50 μm, or 3 to 40 μm. In some embodiments, the depth is substantiallyconstant along the length of the shelf. Alternatively, the depth of theshelf slopes, e.g., downward or upward, from the distal end of theliquid channel to the step region. The final depth of the sloped shelfmay be, for example, from 5% to 1000% greater than the shortest depth,e.g., 10 to 750%, 10 to 500%, 50 to 500%, 60 to 250%, 70 to 200%, or 100to 150%.The overall length of the shelf region may be from at leastabout 0.1 μm to about 1000 μm, e.g., 0.1 to 750 μm, 0.1 to 500 μm, 0.1to 250 μm, 0.1 to 150 μm, 1 to 150 μm, 10 to 150 μm, 50 to 150 μm, 100to 150 μm, 10 to 80 μm, or 10 to 50 μm. In certain embodiments, thelateral walls of the shelf region, i.e., those defining the width, maybe not parallel to one another. In other embodiments, the walls of theshelf region may narrower from the distal end of the first channeltowards the step region. For example, the width of the shelf regionadjacent the distal end of the first channel may be sufficiently largeto support droplet formation. In other embodiments, the shelf region isnot substantially rectangular, e.g., not rectangular or not rectangularwith rounded or chamfered corners.

A step region includes a spatial displacement (e.g., depth). Typically,this displacement occurs at an angle of approximately 90°, e.g., between85° and 95°. Other angles are possible, e.g., 10-90°, e.g., 20 to 90°,45 to 90°, or 70 to 90°. The spatial displacement of the step region maybe any suitable size to be accommodated on a device, as the ultimateextent of displacement does not affect performance of the device.Preferably the displacement is several times the diameter of the dropletbeing formed. In certain embodiments, the displacement is from about 1μm to about 10 cm, e.g., at least 10 μm, at least 40 μm, at least 100μm, or at least 500 μm, e.g., 40 μm to 600 μm. In some cases, the depthof the step region is substantially constant. Alternatively, the depthof the step region may increase away from the shelf region, e.g., toallow droplets that sink or float to roll away from the spatialdisplacement as they are formed. The step region may also increase indepth in two dimensions relative to the shelf region, e.g., both aboveand below the plane of the shelf region. The reservoir may have an inletand/or an outlet for the addition of continuous phase, flow ofcontinuous phase, or removal of the continuous phase and/or droplets.

While dimension of the devices may be described as width or depths, thechannels, shelf regions, and step regions may be disposed in any plane.For example, the width of the shelf may be in the x-y plane, the x-zplane, the y-z plane or any plane therebetween. In addition, a dropletformation region, e.g., including a shelf region, may be laterallyspaced in the x-y plane relative to the first channel or located aboveor below the first channel. Similarly, a droplet formation region, e.g.,including a step region, may be laterally spaced in the x-y plane, e.g.,relative to a shelf region or located above or below a shelf region. Thespatial displacement in a step region may be oriented in any planesuitable to allow the nascent droplet to form a spherical shape. Thefluidic components may also be in different planes so long asconnectivity and other dimensional requirements are met.

The device may also include reservoirs for liquid reagents. For example,the device may include a reservoir for the liquid to flow in the firstchannel and/or a reservoir for the liquid into which droplets areformed. In some cases, devices of the invention include a collectionregion, e.g., a volume for collecting formed droplets. A dropletcollection region may be a reservoir that houses continuous phase or canbe any other suitable structure, e.g., a channel, a shelf, a chamber, ora cavity, on or in the device. For reservoirs or other elements used incollection, the walls may be smooth and not include an orthogonalelement that would impede droplet movement. For example, the walls maynot include any feature that at least in part protrudes or recedes fromthe surface. It will be understood, however, that such elements may havea ceiling or floor. The droplets that are formed may be moved out of thepath of the next droplet being formed by gravity (either upward ordownward depending on the relative density of the droplet and continuousphase). Alternatively or in addition, formed droplets may be moved outof the path of the next droplet being formed by an external forceapplied to the liquid in the collection region, e.g., gentle stirring,flowing continuous phase, or vibration. Similarly, a reservoir forliquids to flow in additional channels, such as those intersecting thefirst channel may be present. A single reservoir may also be connectedto multiple channels in a device, e.g., when the same liquid is to beintroduced at two or more different locations in the device. Wastereservoirs or overflow reservoirs may also be included to collect wasteor overflow when droplets are formed. Alternatively, the device may beconfigured to mate with sources of the liquids, which may be externalreservoirs such as vials, tubes, or pouches. Similarly, the device maybe configured to mate with a separate component that houses thereservoirs. Reservoirs may be of any appropriate size, e.g., to hold 10μL to 500 mL, e.g., 10 μL to 300 mL, 25 μL to 10 mL, 100 μL to 1 mL, 40μL to 300 μL, 1 mL to 10 mL, or 10 mL to 50 mL. When multiple reservoirsare present, each reservoir may have the same or a different size.

In addition to the components discussed above, devices of the inventioncan include additional components. For example, channels may includefilters to prevent introduction of debris into the device. In somecases, the microfluidic systems described herein may comprise one ormore liquid flow units to direct the flow of one or more liquids, suchas the aqueous liquid and/or the second liquid immiscible with theaqueous liquid. In some instances, the liquid flow unit may comprise acompressor to provide positive pressure at an upstream location todirect the liquid from the upstream location to flow to a downstreamlocation. In some instances, the liquid flow unit may comprise a pump toprovide negative pressure at a downstream location to direct the liquidfrom an upstream location to flow to the downstream location. In someinstances, the liquid flow unit may comprise both a compressor and apump, each at different locations. In some instances, the liquid flowunit may comprise different devices at different locations. The liquidflow unit may comprise an actuator. In some instances, where the secondliquid is substantially stationary, the reservoir may maintain aconstant pressure field at or near each droplet formation region.Devices may also include various valves to control the flow of liquidsalong a channel or to allow introduction or removal of liquids ordroplets from the device. Suitable valves are known in the art. Valvesuseful for a device of the present invention include diaphragm valves,solenoid valves, pinch valves, or a combination thereof. Valves can becontrolled manually, electrically, magnetically, hydraulically,pneumatically, or by a combination thereof. The device may also includeintegral liquid pumps or be connectable to a pump to allow for pumpingin the first channels and any other channels requiring flow. Examples ofpressure pumps include syringe, peristaltic, diaphragm pumps, andsources of vacuum. Other pumps can employ centrifugal or electrokineticforces. Alternatively, liquid movement may be controlled by gravity,capillarity, or surface treatments. Multiple pumps and mechanisms forliquid movement may be employed in a single device. The device may alsoinclude one or more vents to allow pressure equalization, and one ormore filters to remove particulates or other undesirable components froma liquid. The device may also include one or more inlets and or outlets,e.g., to introduce liquids and/or remove droplets. Such additionalcomponents may be actuated or monitored by one or more controllers orcomputers operatively coupled to the device, e.g., by being integratedwith, physically connected to (mechanically or electrically), or bywired or wireless connection.

Surface Properties

A surface of the device may include a material, coating, or surfacetexture that determines the physical properties of the device. Inparticular, the flow of liquids through a device of the invention may becontrolled by the device surface properties (e.g., water contact angleof a liquid-contacting surface). In some cases, a device portion (e.g.,a channel or droplet formation region) may have a surface having a watercontact angle suitable for facilitating liquid flow (e.g., in a channel)or assisting droplet formation of a first liquid in a second liquid(e.g., in a droplet formation region).

A device may include a channel having a surface with a first watercontact angle in fluid communication with (e.g., fluidically connectedto) a droplet formation region having a surface with a second watercontact angle. The surface water contact angles may be suited toproducing droplets of a first liquid in a second liquid. In thisnon-limiting example, the channel carrying the first liquid may havesurface with a first water contact angle suited for the first liquidwetting the channel surface. For example, when the first liquid issubstantially miscible with water (e.g., the first liquid is an aqueousliquid), the first water contact angle may be about 95° or less (e.g.,90° or less). Additionally, in this non-limiting example, the dropletformation region may have a surface with a second water contact anglesuited for the second liquid wetting the droplet formation regionsurface (e.g., shelf surface). For example, when the second liquid issubstantially immiscible with water (e.g., the second liquid is an oil),the second water contact angle may be about 70° or more (e.g., 90° ormore, 95° or more, or 100° or more). Typically, in this non-limitingexample, the second water contact angle will differ from the first watercontact angle by 5° to 100°. For example, when the first liquid issubstantially miscible with water (e.g., the first liquid is an aqueousliquid), and the second liquid is substantially immiscible with water(e.g., the second liquid is an oil), the second water contact angle maybe greater than the first water contact angle by 5° to 100°.

For example, portions of the device carrying aqueous phases (e.g., achannel) may have a surface with a water contact angle of less than orequal to about 90° (e.g., include a hydrophilic material or coating),and/or portions of the device housing an oil phase may have a surfacewith a water contact angle of greater than 70° (e.g., greater than 90°,greater than 95°, greater than 100° (e.g., 95°-120° or 100°-110°)),e.g., include a hydrophobic material or coating. In certain embodiments,the droplet formation region may include a material or surface coatingthat reduces or prevents wetting by aqueous phases. For example, thedroplet formation region may have a surface with a water contact angleof greater than 70° (e.g., greater than 90°, greater than 95°, greaterthan 100° (e.g., 95°-120° or 100°-110°)). The device can be designed tohave a single type of material or coating throughout. Alternatively, thedevice may have separate regions having different materials or coatings.Surface textures may also be employed to control fluid flow.

The device surface properties may be those of a native surface (i.e.,the surface properties of the bulk material used for the devicefabrication) or of a surface treatment. Non-limiting examples of surfacetreatments include, e.g., surface coatings and surface textures. In oneapproach, the device surface properties are attributable to one or moresurface coatings present in a device portion. Hydrophobic coatings mayinclude fluoropolymers (e.g., AQUAPEL® glass treatment), silanes,siloxanes, silicones, or other coatings known in the art. Other coatingsinclude those vapor deposited from a precursor such ashenicosyl-1,1,2,2-tetrahydrododecyldimethyltris(dimethylaminosilane);hen icosyl-1,1,2,2-tetrahydrododecyltrichlorosilane (C12);heptadecafluoro-1,1,2,2-tetrahydrodecyltrichlorosilane (C10);nonafluoro-1,1,2,2-tetrahydrohexyltris(dimethylamino)silane;3,3,3,4,4,5,5,6,6-nonafluorohexyltrichlorosilane;tridecafluoro-1,1,2,2-tetrahydrooctyltrichlorosilane (C8);bis(tridecafluoro-1,1,2,2-tetrahydrooctyl)dimethylsiloxymethylchlorosilane;nonafluorohexyltriethoxysilane (C6); dodecyltrichlorosilane (DTS);dimethyldichlorosilane (DDMS); or 10-undecenyltrichlorosilane (V11);pentafluorophenylpropyltrichlorosilane (C5). Hydrophilic coatingsinclude polymers such as polysaccharides, polyethylene glycol,polyamines, and polycarboxylic acids. Hydrophilic surfaces may also becreated by oxygen plasma treatment of certain materials.

A coated surface may be formed by depositing a metal oxide onto asurface of the device. Example metal oxides useful for coating surfacesinclude, but are not limited to, Al₂O₃, TiO₂, SiO₂, or a combinationthereof. Other metal oxides useful for surface modifications are knownin the art. The metal oxide can be deposited onto a surface by standarddeposition techniques, including, but not limited to, atomic layerdeposition (ALD), physical vapor deposition (PVD), e.g., sputtering,chemical vapor deposition (CVD), or laser deposition. Other depositiontechniques for coating surfaces, e.g., liquid-based deposition, areknown in the art. For example, an atomic layer of Al₂O₃ can be depositedon a surface by contacting it with trimethylaluminum (TMA) and water.

In another approach, the device surface properties may be attributableto surface texture. For example, a surface may have a nanotexture, e.g.,have a surface with nanometer surface features, such as cones orcolumns, that alters the wettability of the surface. Nanotexturedsurface may be hydrophilic, hydrophobic, or superhydrophobic, e.g., havea water contact angle greater than 150°. Exemplary superhydrophobicmaterials include Manganese Oxide Polystyrene (MnO₂/PS) nano-composite,Zinc Oxide Polystyrene (ZnO/PS) nano-composite, Precipitated CalciumCarbonate, Carbon nano-tube structures, and a silica nano-coating.Superhydrophobic coatings may also include a low surface energy material(e.g., an inherently hydrophobic material) and a surface roughness(e.g., using laser ablation techniques, plasma etching techniques, orlithographic techniques in which a material is etched through aperturesin a patterned mask). Examples of low surface energy materials includefluorocarbon materials, e.g., polytetrafluoroethylene (PTFE),fluorinated ethylene propylene (FEP), ethylene tetrafluoroethylene(ETFE), ethylene chloro-trifluoroethylene (ECTFE), perfluoro-alkoxyalkane (PFA), poly(chloro-trifluoro-ethylene) (CTFE),perfluoro-alkoxyalkane (PFA), and poly(vinylidene fluoride) (PVDF).Other superhydrophobic surfaces are known in the art.

In some cases, the first water contact angle is less than or equal toabout 90°, e.g., less than 80°, 70°, 60°, 50°, 40°, 30°, 20°, or 10°,e.g., 90°, 85°, 80°, 75°, 70°, 65°, 60°, 55°, 50°, 45°, 40°, 35°, 30°,25°, 20°, 15°, 10°, 9°, 8°, 7°, 6°, 5°, 4°, 3°, 2°, 1°, or 0°. In somecases, the second water contact angle is at least 70°, e.g., at least80°, at least 85°, at least 90°, at least 95°, or at least 100° (e.g.,about 100°, 101°, 102°, 103°, 104°, 105°, 106°, 107°, 108°, 109°, 110°,115°, 120°, 125°, 130°, 135°, 140°, 145°, or about 150°.

The difference between the first and second water contact angles may be5° to 100°, e.g., 5° to 80°, 5° to 60°, 5° to 50°, 5° to 40°, 5° to 30°,5° to 20°, 10° to 75°, 15° to 70°, 20° to 65°, 25° to 60°, 30 to 50°,35° to 45°, e.g., 5°, 6°, 7°, 8°, 9°, 10°, 15°, 20°, 25°, 30°, 35°, 40°,45°, 50°, 55°, 60, 65°, 70°, 75°, 80°, 85°, 90°, 95°, or 100°.

The above discussion centers on the water contact angle. It will beunderstood that liquids employed in the devices and methods of theinvention may not be water, or even aqueous. Accordingly, the actualcontact angle of a liquid on a surface of the device may differ from thewater contact angle.

Particles

The invention includes devices, systems, and kits having particles,e.g., for use in analyte detection. For example, particles configuredwith analyte detection moieties (e.g., barcodes, nucleic acids, bindingmolecules (e.g., proteins, peptides, aptamers, antibodies, or antibodyfragments), enzymes, substrates, etc.) can be included in a dropletcontaining an analyte to modify the analyte and/or detect the presenceor concentration of the analyte. In some embodiments, particles aresynthetic particles (e.g., beads, e.g., gel beads).

For example, a droplet may include one or more analyte-detectionmoieties, e.g., unique identifiers, such as barcodes. Analyte-detectionmoieties, e.g., barcodes, may be introduced into droplets previous to,subsequent to, or concurrently with droplet formation. The delivery ofthe analyte-detection moieties, e.g., barcodes, to a particular dropletallows for the later attribution of the characteristics of an individualsample (e.g., biological particle) to the particular droplet.Analyte-detection moieties, e.g., barcodes, may be delivered, forexample on a nucleic acid (e.g., an oligonucleotide), to a droplet viaany suitable mechanism. Analyte-detection moieties, e.g., barcodednucleic acids (e.g., oligonucleotides), can be introduced into a dropletvia a particle, such as a microcapsule. In some cases, analyte-detectionmoieties, e.g., barcoded nucleic acids (e.g., oligonucleotides), can beinitially associated with the particle (e.g., microcapsule) and thenreleased upon application of a stimulus which allows theanalyte-detection moieties, e.g., nucleic acids (e.g.,oligonucleotides), to dissociate or to be released from the particle.

A particle, e.g., a bead, may be porous, non-porous, hollow (e.g., amicrocapsule), solid, semi-solid, semi-fluidic, fluidic, and/or acombination thereof. In some instances, a particle, e.g., a bead, may bedissolvable, disruptable, and/or degradable. In some cases, a particle,e.g., a bead, may not be degradable. In some cases, the particle, e.g.,a bead, may be a gel bead. A gel bead may be a hydrogel bead. A gel beadmay be formed from molecular precursors, such as a polymeric ormonomeric species. A semi-solid particle, e.g., a bead, may be aliposomal bead. Solid particles, e.g., beads, may comprise metalsincluding iron oxide, gold, and silver. In some cases, the particle,e.g., the bead, may be a silica bead. In some cases, the particle, e.g.,a bead, can be rigid. In other cases, the particle, e.g., a bead, may beflexible and/or compressible.

A particle, e.g., a bead, may comprise natural and/or syntheticmaterials. For example, a particle, e.g., a bead, can comprise a naturalpolymer, a synthetic polymer or both natural and synthetic polymers.Examples of natural polymers include proteins and sugars such asdeoxyribonucleic acid, rubber, cellulose, starch (e.g., amylose,amylopectin), proteins, enzymes, polysaccharides, silks,polyhydroxyalkanoates, chitosan, dextran, collagen, carrageenan,ispaghula, acacia, agar, gelatin, shellac, sterculia gum, xanthan gum,Corn sugar gum, guar gum, gum karaya, agarose, alginic acid, alginate,or natural polymers thereof. Examples of synthetic polymers includeacrylics, nylons, silicones, spandex, viscose rayon, polycarboxylicacids, polyvinyl acetate, polyacrylamide, polyacrylate, polyethyleneglycol, polyurethanes, polylactic acid, silica, polystyrene,polyacrylonitrile, polybutadiene, polycarbonate, polyethylene,polyethylene terephthalate, poly(chlorotrifluoroethylene), poly(ethyleneoxide), poly(ethylene terephthalate), polyethylene, polyisobutylene,poly(methyl methacrylate), poly(oxymethylene), polyformaldehyde,polypropylene, polystyrene, poly(tetrafluoroethylene), poly(vinylacetate), poly(vinyl alcohol), poly(vinyl chloride), poly(vinylidenedichloride), poly(vinylidene difluoride), poly(vinyl fluoride) and/orcombinations (e.g., co-polymers) thereof. Beads may also be formed frommaterials other than polymers, including lipids, micelles, ceramics,glass-ceramics, material composites, metals, other inorganic materials,and others.

In some instances, the particle, e.g., the bead, may contain molecularprecursors (e.g., monomers or polymers), which may form a polymernetwork via polymerization of the molecular precursors. In some cases, aprecursor may be an already polymerized species capable of undergoingfurther polymerization via, for example, a chemical cross-linkage. Insome cases, a precursor can comprise one or more of an acrylamide or amethacrylamide monomer, oligomer, or polymer. In some cases, theparticle, e.g., the bead, may comprise prepolymers, which are oligomerscapable of further polymerization. For example, polyurethane beads maybe prepared using prepolymers. In some cases, the particle, e.g., thebead, may contain individual polymers that may be further polymerizedtogether. In some cases, particles, e.g., beads, may be generated viapolymerization of different precursors, such that they comprise mixedpolymers, co-polymers, and/or block co-polymers. In some cases, theparticle, e.g., the bead, may comprise covalent or ionic bonds betweenpolymeric precursors (e.g., monomers, oligomers, linear polymers),oligonucleotides, primers, and other entities. In some cases, thecovalent bonds can be carbon-carbon bonds or thioether bonds.

Cross-linking may be permanent or reversible, depending upon theparticular cross-linker used. Reversible cross-linking may allow for thepolymer to linearize or dissociate under appropriate conditions. In somecases, reversible cross-linking may also allow for reversible attachmentof a material bound to the surface of a bead. In some cases, across-linker may form disulfide linkages. In some cases, the chemicalcross-linker forming disulfide linkages may be cystamine or a modifiedcystamine.

Particles, e.g., beads, may be of uniform size or heterogeneous size. Insome cases, the diameter of a particle, e.g., a bead, may be at leastabout 1 micrometer (μm), 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm,70 μm, 80 μm, 90 μm, 100 μm, 250 μm, 500 μm, 1 mm, or greater. In somecases, a particle, e.g., a bead, may have a diameter of less than about1 μm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90μm, 100 μm, 250 μm, 500 μm, 1 mm, or less. In some cases, a particle,e.g., a bead, may have a diameter in the range of about 40-75 μm, 30-75μm, 20-75 μm, 40-85 μm, 40-95 μm, 20-100 μm, 10-100 μm, 1-100 μm, 20-250μm, or 20-500 μm. The size of a particle, e.g., a bead, e.g., a gelbead, used to produce droplets is typically on the order of a crosssection of the first channel (width or depth). In some cases, the gelbeads are larger than the width and/or depth of the first channel and/orshelf, e.g., at least 1.5×, 2×, 3×, or 4× larger than the width and/ordepth of the first channel and/or shelf.

In certain embodiments, particles, e.g., beads, can be provided as apopulation or plurality of particles, e.g., beads, having a relativelymonodisperse size distribution. Where it may be desirable to providerelatively consistent amounts of reagents within droplets, maintainingrelatively consistent particle, e.g., bead, characteristics, such assize, can contribute to the overall consistency. In particular, theparticles, e.g., beads, described herein may have size distributionsthat have a coefficient of variation in their cross-sectional dimensionsof less than 50%, less than 40%, less than 30%, less than 20%, and insome cases less than 15%, less than 10%, less than 5%, or less.

Particles may be of any suitable shape. Examples of particles, e.g.,beads, shapes include, but are not limited to, spherical, non-spherical,oval, oblong, amorphous, circular, cylindrical, and variations thereof.

A particle, e.g., bead, injected or otherwise introduced into a dropletmay comprise releasably, cleavably, or reversibly attached analytedetection moieties (e.g., barcodes). A particle, e.g., bead, injected orotherwise introduced into a droplet may comprise activatable analytedetection moieties (e.g., barcodes).

A particle, e.g., bead, injected or otherwise introduced into a dropletmay be a degradable, disruptable, or dissolvable particle, e.g.,dissolvable bead.

Particles, e.g., beads, within a channel may flow at a substantiallyregular flow profile (e.g., at a regular flow rate). Such regular flowprofiles can permit a droplet, when formed, to include a single particle(e.g., bead) and a single cell or other biological particle. Suchregular flow profiles may permit the droplets to have an dual occupancy(e.g., droplets having at least one bead and at least one cell or otherbiological particle) greater than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97% 98%, or 99% of the population. In some embodiments,the droplets have a 1:1 dual occupancy (i.e., droplets having exactlyone particle (e.g., bead) and exactly one cell or other biologicalparticle) greater than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97% 98%, or 99% of the population. Such regular flow profiles anddevices that may be used to provide such regular flow profiles areprovided, for example, in U.S. Patent Publication No. 2015/0292988,which is entirely incorporated herein by reference.

As discussed above, analyte-detection moieties (e.g., barcodes) can bereleasably, cleavably or reversibly attached to the particles, e.g.,beads, such that analyte detection moieties (e.g., barcodes) can bereleased or be releasable through cleavage of a linkage between thebarcode molecule and the particle, e.g., bead, or released throughdegradation of the particle (e.g., bead) itself, allowing the barcodesto be accessed or be accessible by other reagents, or both. Releasableanalyte-detection moieties (e.g., barcodes) may sometimes be referred toas activatable analyte-detection moieties (e.g., activatable barcodes),in that they are available for reaction once released. Thus, forexample, an activatable analyte detection-moiety (e.g., activatablebarcode) may be activated by releasing the analyte detection moiety(e.g., barcode) from a particle, e.g., bead (or other suitable type ofdroplet described herein). Other activatable configurations are alsoenvisioned in the context of the described methods and systems.

In addition to, or as an alternative to the cleavable linkages betweenthe particles, e.g., beads, and the associated antigen detectionmoieties, such as barcode containing nucleic acids (e.g.,oligonucleotides), the particles, e.g., beads may be degradable,disruptable, or dissolvable spontaneously or upon exposure to one ormore stimuli (e.g., temperature changes, pH changes, exposure toparticular chemical species or phase, exposure to light, reducing agent,etc.). In some cases, a particle, e.g., bead, may be dissolvable, suchthat material components of the particle, e.g., bead, are degraded orsolubilized when exposed to a particular chemical species or anenvironmental change, such as a change temperature or a change in pH. Insome cases, a gel bead can be degraded or dissolved at elevatedtemperature and/or in basic conditions. In some cases, a particle, e.g.,bead, may be thermally degradable such that when the particle, e.g.,bead, is exposed to an appropriate change in temperature (e.g., heat),the particle, e.g., bead, degrades. Degradation or dissolution of aparticle (e.g., bead) bound to a species (e.g., a nucleic acid, e.g., anoligonucleotide, e.g., barcoded oligonucleotide) may result in releaseof the species from the particle, e.g., bead. As will be appreciatedfrom the above disclosure, the degradation of a particle, e.g., bead,may refer to the disassociation of a bound or entrained species from aparticle, e.g., bead, both with and without structurally degrading thephysical particle, e.g., bead, itself. For example, entrained speciesmay be released from particles, e.g., beads, through osmotic pressuredifferences due to, for example, changing chemical environments. By wayof example, alteration of particle, e.g., bead, pore sizes due toosmotic pressure differences can generally occur without structuraldegradation of the particle, e.g., bead, itself. In some cases, anincrease in pore size due to osmotic swelling of a particle, e.g., beador microcapsule (e.g., liposome), can permit the release of entrainedspecies within the particle. In other cases, osmotic shrinking of aparticle may cause the particle, e.g., bead, to better retain anentrained species due to pore size contraction.

A degradable particle, e.g., bead, may be introduced into a droplet,such as a droplet of an emulsion or a well, such that the particle,e.g., bead, degrades within the droplet and any associated species(e.g., nucleic acids, oligonucleotides, or fragments thereof) arereleased within the droplet when the appropriate stimulus is applied.The free species (e.g., nucleic acid, oligonucleotide, or fragmentthereof) may interact with other reagents contained in the droplet. Forexample, a polyacrylamide bead comprising cystamine and linked, via adisulfide bond, to a barcode sequence, may be combined with a reducingagent within a droplet of a water-in-oil emulsion. Within the droplet,the reducing agent can break the various disulfide bonds, resulting inparticle, e.g., bead, degradation and release of the barcode sequenceinto the aqueous, inner environment of the droplet. In another example,heating of a droplet comprising a particle-, e.g., bead-, boundanalyte-detection moiety (e.g., barcode) in basic solution may alsoresult in particle, e.g., bead, degradation and release of the attachedbarcode sequence into the aqueous, inner environment of the droplet.

Any suitable number of analyte-detection moieties (e.g., molecular tagmolecules (e.g., primer, barcoded oligonucleotide, etc.)) can beassociated with a particle, e.g., bead, such that, upon release from theparticle, the analyte detection moieties (e.g., molecular tag molecules(e.g., primer, e.g., barcoded oligonucleotide, etc.)) are present in thedroplet at a pre-defined concentration. Such pre-defined concentrationmay be selected to facilitate certain reactions for generating asequencing library, e.g., amplification, within the droplet. In somecases, the pre-defined concentration of a primer can be limited by theprocess of producing oligonucleotide-bearing particles, e.g., beads.

Additional reagents may be included as part of the particles (e.g.,analyte-detection moieties) and/or in solution or dispersed in thedroplet, for example, to activate, mediate, or otherwise participate ina reaction, e.g., between the analyte and analyte-detection moiety.

Biological Samples

A droplet of the present disclosure may include biological particles(e.g., cells) and/or macromolecular constituents thereof (e.g.,components of cells (e.g., intracellular or extracellular proteins,nucleic acids, glycans, or lipids) or products of cells (e.g., secretionproducts)). An analyte from a biological particle, e.g., component orproduct thereof, may be considered to be a bioanalyte. In someembodiments, a biological particle, e.g., cell, or product thereof isincluded in a droplet, e.g., with one or more particles (e.g., beads)having an analyte detection moiety. A biological particle, e.g., cell,and/or components or products thereof can, in some embodiments, beencased inside a gel, such as via polymerization of a droplet containingthe biological particle and precursors capable of being polymerized orgelled.

In the case of encapsulated biological particles (e.g., cells), abiological particle may be included in a droplet that contains lysisreagents in order to release the contents (e.g., contents containing oneor more analytes (e.g., bioanalytes)) of the biological particles withinthe droplet. In such cases, the lysis agents can be contacted with thebiological particle suspension concurrently with, or immediately priorto the introduction of the biological particles into the dropletformation region, for example, through an additional channel or channelsupstream or proximal to a second channel or a third channel that isupstream or proximal to a second droplet formation region. Examples oflysis agents include bioactive reagents, such as lysis enzymes that areused for lysis of different cell types, e.g., gram positive or negativebacteria, plants, yeast, mammalian, etc., such as lysozymes,achromopeptidase, lysostaphin, labiase, kitalase, lyticase, and avariety of other lysis enzymes available from, e.g., Sigma-Aldrich, Inc.(St Louis, Mo.), as well as other commercially available lysis enzymes.Other lysis agents may additionally or alternatively be contained in adroplet with the biological particles (e.g., cells) to cause the releaseof the biological particles' contents into the droplets. For example, insome cases, surfactant based lysis solutions may be used to lyse cells,although these may be less desirable for emulsion based systems wherethe surfactants can interfere with stable emulsions. In some cases,lysis solutions may include non-ionic surfactants such as, for example,TritonX-100 and Tween 20. In some cases, lysis solutions may includeionic surfactants such as, for example, sarcosyl and sodium dodecylsulfate (SDS). In some embodiments, lysis solutions are hypotonic,thereby lysing cells by osmotic shock. Electroporation, thermal,acoustic or mechanical cellular disruption may also be used in certaincases, e.g., non-emulsion based droplet formation such as encapsulationof biological particles that may be in addition to or in place ofdroplet formation, where any pore size of the encapsulate issufficiently small to retain nucleic acid fragments of a desired size,following cellular disruption.

In addition to the lysis agents, other reagents can also be included indroplets with the biological particles, including, for example, DNaseand RNase inactivating agents or inhibitors, such as proteinase K,chelating agents, such as EDTA, and other reagents employed in removingor otherwise reducing negative activity or impact of different celllysate components on subsequent processing of nucleic acids. Inaddition, in the case of encapsulated biological particles (e.g.,cells), the biological particles may be exposed to an appropriatestimulus to release the biological particles or their contents from amicrocapsule within a droplet. For example, in some cases, a chemicalstimulus may be included in a droplet along with an encapsulatedbiological particle to allow for degradation of the encapsulating matrixand release of the cell or its contents into the larger droplet. In somecases, this stimulus may be the same as the stimulus described elsewhereherein for release of analyte detection moieties (e.g.,oligonucleotides) from their respective particle (e.g., bead). Inalternative aspects, this may be a different and non-overlappingstimulus, in order to allow an encapsulated biological particle to bereleased into a droplet at a different time from the release of analytedetection moieties (e.g., oligonucleotides) into the same droplet.

Additional reagents may also be included in droplets with the biologicalparticles, such as endonucleases to fragment a biological particle'sDNA, DNA polymerase enzymes and dNTPs used to amplify the biologicalparticle's nucleic acid fragments and to attach the barcode moleculartags to the amplified fragments. Other reagents may also include reversetranscriptase enzymes, including enzymes with terminal transferaseactivity, primers and oligonucleotides, and switch oligonucleotides(also referred to herein as “switch oligos” or “template switchingoligonucleotides”) which can be used for template switching. In somecases, template switching can be used to increase the length of a cDNA.In some cases, template switching can be used to append a predefinednucleic acid sequence to the cDNA. In an example of template switching,cDNA can be generated from reverse transcription of a template, e.g.,cellular mRNA, where a reverse transcriptase with terminal transferaseactivity can add additional nucleotides, e.g., polyC, to the cDNA in atemplate independent manner. Switch oligos can include sequencescomplementary to the additional nucleotides, e.g., polyG. The additionalnucleotides (e.g., polyC) on the cDNA can hybridize to the additionalnucleotides (e.g., polyG) on the switch oligo, whereby the switch oligocan be used by the reverse transcriptase as template to further extendthe cDNA. Template switching oligonucleotides may comprise ahybridization region and a template region. The hybridization region cancomprise any sequence capable of hybridizing to the target. In somecases, as previously described, the hybridization region comprises aseries of G bases to complement the overhanging C bases at the 3′ end ofa cDNA molecule. The series of G bases may comprise 1 G base, 2 G bases,3 G bases, 4 G bases, 5 G bases or more than 5 G bases. The templatesequence can comprise any sequence to be incorporated into the cDNA. Insome cases, the template region comprises at least 1 (e.g., at least 2,3, 4, 5 or more) tag sequences and/or functional sequences. Switcholigos may comprise deoxyribonucleic acids; ribonucleic acids; modifiednucleic acids including 2-Aminopurine, 2,6-Diaminopurine (2-Amino-dA),inverted dT, 5-Methyl dC, 2′-deoxyinosine, Super T(5-hydroxybutynl-2′-deoxyuridine), Super G (8-aza-7-deazaguanosine),locked nucleic acids (LNAs), unlocked nucleic acids (UNAs, e.g., UNA-A,UNA-U, UNA-C, UNA-G), Iso-dG, Iso-dC, 2′ Fluoro bases (e.g., Fluoro C,Fluoro U, Fluoro A, and Fluoro G), or any combination.

In some cases, the length of a switch oligo may be 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182,183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196,197 , 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210,211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224,225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238,239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250 nucleotidesor longer.

In some cases, the length of a switch oligo may be at least 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139,140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153,154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167,168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181,182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195,196, 197 , 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209,210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223,224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237,238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249 or 250nucleotides or longer.

In some cases, the length of a switch oligo may be at most 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139,140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153,154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167,168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181,182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195,196, 197 , 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209,210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223,224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237,238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249 or 250nucleotides.

Once the contents of the cells are released into their respectivedroplets, the macromolecular components (e.g., macromolecularconstituents of biological particles, such as RNA, DNA, or proteins)contained therein may be further processed within the droplets.

As described above, the macromolecular components (e.g., bioanalytes) ofindividual biological particles (e.g., cells) can be provided withunique identifiers (e.g., barcodes) such that upon characterization ofthose macromolecular components, at which point components from aheterogeneous population of cells may have been mixed and areinterspersed or solubilized in a common liquid, any given component(e.g., bioanalyte) may be traced to the biological particle (e.g., cell)from which it was obtained. The ability to attribute characteristics toindividual biological particles or groups of biological particles isprovided by the assignment of unique identifiers specifically to anindividual biological particle or groups of biological particles. Uniqueidentifiers, for example, in the form of nucleic acid barcodes, can beassigned or associated with individual biological particles (e.g.,cells) or populations of biological particles (e.g., cells), in order totag or label the biological particle's macromolecular components (and asa result, its characteristics) with the unique identifiers. These uniqueidentifiers can then be used to attribute the biological particle'scomponents and characteristics to an individual biological particle orgroup of biological particles. This can be performed by forming dropletsincluding the individual biological particle or groups of biologicalparticles with the unique identifiers (via particles, e.g., beads), asdescribed in the systems and methods herein.

In some aspects, the unique identifiers are provided in the form ofoligonucleotides that comprise nucleic acid barcode sequences that maybe attached to or otherwise associated with the nucleic acid contents ofindividual biological particle, or to other components of the biologicalparticle, and particularly to fragments of those nucleic acids. Theoligonucleotides are partitioned such that as between oligonucleotidesin a given droplet, the nucleic acid barcode sequences contained thereinare the same, but as between different droplets, the oligonucleotidescan, and do have differing barcode sequences, or at least represent alarge number of different barcode sequences across all of the dropletsin a given analysis. In some aspects, only one nucleic acid barcodesequence can be associated with a given droplet, although in some cases,two or more different barcode sequences may be present.

The nucleic acid barcode sequences can include from 6 to about 20 ormore nucleotides within the sequence of the oligonucleotides. In somecases, the length of a barcode sequence may be 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In some cases, thelength of a barcode sequence may be at least 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In some cases, thelength of a barcode sequence may be at most 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20 nucleotides or shorter. These nucleotides maybe completely contiguous, i.e., in a single stretch of adjacentnucleotides, or they may be separated into two or more separatesubsequences that are separated by 1 or more nucleotides. In some cases,separated barcode subsequences can be from about 4 to about 16nucleotides in length. In some cases, the barcode subsequence may be 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In somecases, the barcode subsequence may be at least 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16 nucleotides or longer. In some cases, the barcodesubsequence may be at most 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16nucleotides or shorter.

Analyte-detection moieties (e.g., oligonucleotides) in droplets can alsoinclude other functional sequences useful in processing of nucleic acidsfrom biological particles contained in the droplet. These sequencesinclude, for example, targeted or random/universal amplification primersequences for amplifying the genomic DNA from the individual biologicalparticles within the droplets while attaching the associated barcodesequences, sequencing primers or primer recognition sites, hybridizationor probing sequences, e.g., for identification of presence of thesequences or for pulling down barcoded nucleic acids, or any of a numberof other potential functional sequences.

Other mechanisms of forming droplets containing oligonucleotides mayalso be employed, including, e.g., coalescence of two or more droplets,where one droplet contains oligonucleotides, or microdispensing ofoligonucleotides into droplets, e.g., droplets within microfluidicsystems.

In an example, particles (e.g., beads) are provided that each includelarge numbers of the above described barcoded oligonucleotidesreleasably attached to the beads, where all of the oligonucleotidesattached to a particular bead will include the same nucleic acid barcodesequence, but where a large number of diverse barcode sequences arerepresented across the population of beads used. In some embodiments,hydrogel beads, e.g., beads having polyacrylamide polymer matrices, areused as a solid support and delivery vehicle for the oligonucleotidesinto the droplets, as they are capable of carrying large numbers ofoligonucleotide molecules, and may be configured to release thoseoligonucleotides upon exposure to a particular stimulus, as describedelsewhere herein. In some cases, the population of beads will provide adiverse barcode sequence library that includes at least about 1,000different barcode sequences, at least about 5,000 different barcodesequences, at least about 10,000 different barcode sequences, at leastabout 50,000 different barcode sequences, at least about 100,000different barcode sequences, at least about 1,000,000 different barcodesequences, at least about 5,000,000 different barcode sequences, or atleast about 10,000,000 different barcode sequences, or more.Additionally, each bead can be provided with large numbers ofoligonucleotide molecules attached. In particular, the number ofmolecules of oligonucleotides including the barcode sequence on anindividual bead can be at least about 1,000 oligonucleotide molecules,at least about 5,000 oligonucleotide molecules, at least about 10,000oligonucleotide molecules, at least about 50,000 oligonucleotidemolecules, at least about 100,000 oligonucleotide molecules, at leastabout 500,000 oligonucleotides, at least about 1,000,000 oligonucleotidemolecules, at least about 5,000,000 oligonucleotide molecules, at leastabout 10,000,000 oligonucleotide molecules, at least about 50,000,000oligonucleotide molecules, at least about 100,000,000 oligonucleotidemolecules, and in some cases at least about 1 billion oligonucleotidemolecules, or more.

Moreover, when the population of beads are included in droplets, theresulting population of droplets can also include a diverse barcodelibrary that includes at least about 1,000 different barcode sequences,at least about 5,000 different barcode sequences, at least about 10,000different barcode sequences, at least at least about 50,000 differentbarcode sequences, at least about 100,000 different barcode sequences,at least about 1,000,000 different barcode sequences, at least about5,000,000 different barcode sequences, or at least about 10,000,000different barcode sequences. Additionally, each droplet of thepopulation can include at least about 1,000 oligonucleotide molecules,at least about 5,000 oligonucleotide molecules, at least about 10,000oligonucleotide molecules, at least about 50,000 oligonucleotidemolecules, at least about 100,000 oligonucleotide molecules, at leastabout 500,000 oligonucleotides, at least about 1,000,000 oligonucleotidemolecules, at least about 5,000,000 oligonucleotide molecules, at leastabout 10,000,000 oligonucleotide molecules, at least about 50,000,000oligonucleotide molecules, at least about 100,000,000 oligonucleotidemolecules, and in some cases at least about 1 billion oligonucleotidemolecules.

In some cases, it may be desirable to incorporate multiple differentbarcodes within a given droplet, either attached to a single or multipleparticles, e.g., beads, within the droplet. For example, in some cases,mixed, but known barcode sequences set may provide greater assurance ofidentification in the subsequent processing, for example, by providing astronger address or attribution of the barcodes to a given droplet, as aduplicate or independent confirmation of the output from a givendroplet.

Oligonucleotides may be releasable from the particles (e.g., beads) uponthe application of a particular stimulus. In some cases, the stimulusmay be a photo-stimulus, e.g., through cleavage of a photo-labilelinkage that releases the oligonucleotides. In other cases, a thermalstimulus may be used, where increase in temperature of the particle,e.g., bead, environment will result in cleavage of a linkage or otherrelease of the oligonucleotides form the particles, e.g., beads. Instill other cases, a chemical stimulus is used that cleaves a linkage ofthe oligonucleotides to the beads, or otherwise results in release ofthe oligonucleotides from the particles, e.g., beads. In one case, suchcompositions include the polyacrylamide matrices described above forencapsulation of biological particles, and may be degraded for releaseof the attached oligonucleotides through exposure to a reducing agent,such as dithiothreitol (DTT).

The droplets described herein may contain either one or more biologicalparticles (e.g., cells), either one or more barcode carrying particles,e.g., beads, or both at least a biological particle and at least abarcode carrying particle, e.g., bead. In some instances, a droplet maybe unoccupied and contain neither biological particles norbarcode-carrying particles, e.g., beads. As noted previously, bycontrolling the flow characteristics of each of the liquids combining atthe droplet formation region(s), as well as controlling the geometry ofthe droplet formation region(s), droplet formation can be optimized toachieve a desired occupancy level of particles, e.g., beads, biologicalparticles, or both, within the droplets that are generated.

Kits and Systems

Devices of the invention may be combined with various externalcomponents, e.g., pumps, reservoirs, or controllers, reagents, e.g.,analyte detection moieties, liquids, particles (e.g., beads), and/orsample in the form of kits and systems.

Methods

The methods described herein to generate droplets, e.g., of uniform andpredictable sizes, and with high throughput, may be used to greatlyincrease the efficiency of single cell applications and/or otherapplications receiving droplet-based input. Such single cellapplications and other applications may often be capable of processing acertain range of droplet sizes. The methods may be employed to generatedroplets for use as microscale chemical reactors, where the volumes ofthe chemical reactants are small (-pLs).

The methods disclosed herein may produce emulsions, generally, i.e.,droplet of a dispersed phases in a continuous phase. For example,droplets may include a first liquid, and the other liquid may be asecond liquid. The first liquid may be substantially immiscible with thesecond liquid. In some instances, the first liquid may be an aqueousliquid or may be substantially miscible with water. Droplets producedaccording to the methods disclosed herein may combine multiple liquids.For example, a droplet may combine a first and third liquids. The firstliquid may be substantially miscible with the third liquid. The secondliquid may be an oil, as described herein.

A variety of applications require the evaluation of the presence andquantification of different biological particle or organism types withina population of biological particles, including, for example, microbiomeanalysis and characterization, environmental testing, food safetytesting, epidemiological analysis, e.g., in tracing contamination or thelike.

The methods described herein may allow for the production of one or moredroplets containing a single particle, e.g., bead, and/or singlebiological particle (e.g., cell) with uniform and predictable dropletsize. The methods also allow for the production of one or more dropletscomprising a single biological particle (e.g., cell) and more than oneparticle, e.g., bead, one or more droplets comprising more than onebiological particle (e.g., cell) and a single particle, e.g., bead,and/or one or more droplets comprising more than one biological particle(e.g., cell) and more than one particle, e.g., beads. The methods mayalso allow for increased throughput of droplet formation.

Droplets are in general formed by allowing a first liquid to flow into asecond liquid in a droplet formation region, where dropletsspontaneously form as described herein. The droplets may comprise anaqueous liquid dispersed phase within a non-aqueous continuous phase,such as an oil phase. In some cases, droplet formation may occur in theabsence of externally driven movement of the continuous phase, e.g., asecond liquid, e.g., an oil. As discussed above, the continuous phasemay nonetheless be externally driven, even though it is not required fordroplet formation. Emulsion systems for creating stable droplets innon-aqueous (e.g., oil) continuous phases are described in detail in,for example, U.S. Pat. No. 9,012,390, which is entirely incorporatedherein by reference for all purposes. Alternatively or in addition, thedroplets may comprise, for example, micro-vesicles that have an outerbarrier surrounding an inner liquid center or core. In some cases, thedroplets may comprise a porous matrix that is capable of entrainingand/or retaining materials within its matrix. A variety of differentvessels are described in, for example, U.S. Patent ApplicationPublication No. 2014/0155295, which is entirely incorporated herein byreference for all purposes. The droplets can be collected in asubstantially stationary volume of liquid, e.g., with the buoyancy ofthe formed droplets moving them out of the path of nascent droplets (upor down depending on the relative density of the droplets and continuousphase). Alternatively or in addition, the formed droplets can be movedout of the path of nascent droplets actively, e.g.,using a gentle flowof the continuous phase, e.g., a liquid stream or gently stirred liquid.

Allocating particles, e.g., beads (e.g., microcapsules carrying barcodedoligonucleotides) or biological particles (e.g., cells) to discretedroplets may generally be accomplished by introducing a flowing streamof particles, e.g., beads, in an aqueous liquid into a flowing stream ornon-flowing reservoir of a non-aqueous liquid, such that droplets aregenerated. In some instances, the occupancy of the resulting droplets(e.g., number of particles, e.g., beads, per droplet) can be controlledby providing the aqueous stream at a certain concentration or frequencyof particles, e.g., beads. In some instances, the occupancy of theresulting droplets can also be controlled by adjusting one or moregeometric features at the point of droplet formation, such as a width ofa fluidic channel carrying the particles, e.g., beads, relative to adiameter of a given particles, e.g., beads.

Where single particle-, e.g., bead-, containing droplets are desired,the relative flow rates of the liquids can be selected such that, onaverage, the droplets contain fewer than one particle, e.g., bead, perdroplet in order to ensure that those droplets that are occupied areprimarily singly occupied. In some embodiments, the relative flow ratesof the liquids can be selected such that a majority of droplets areoccupied, for example, allowing for only a small percentage ofunoccupied droplets. The flows and channel architectures can becontrolled as to ensure a desired number of singly occupied droplets,less than a certain level of unoccupied droplets and/or less than acertain level of multiply occupied droplets.

The methods described herein can be operated such that a majority ofoccupied droplets include no more than one biological particle peroccupied droplet. In some cases, the droplet formation process isconducted such that fewer than 25% of the occupied droplets contain morethan one biological particle (e.g., multiply occupied droplets), and inmany cases, fewer than 20% of the occupied droplets have more than onebiological particle. In some cases, fewer than 10% or even fewer than 5%of the occupied droplets include more than one biological particle perdroplet.

It may be desirable to avoid the creation of excessive numbers of emptydroplets, for example, from a cost perspective and/or efficiencyperspective. However, while this may be accomplished by providingsufficient numbers of particles, e.g., beads, into the droplet formationregion, the Poisson distribution may expectedly increase the number ofdroplets that may include multiple biological particles. As such, atmost about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%,35%, 30%, 25%, 20%, 15%, 10%, 5% or less of the generated droplets canbe unoccupied. In some cases, the flow of one or more of the particles,or liquids directed into the droplet formation region can be conductedsuch that, in many cases, no more than about 50% of the generateddroplets, no more than about 25% of the generated droplets, or no morethan about 10% of the generated droplets are unoccupied. These flows canbe controlled so as to present non-Poisson distribution of singlyoccupied droplets while providing lower levels of unoccupied droplets.The above noted ranges of unoccupied droplets can be achieved whilestill providing any of the single occupancy rates described above. Forexample, in many cases, the use of the systems and methods describedherein creates resulting droplets that have multiple occupancy rates ofless than about 25%, less than about 20%, less than about 15%, less thanabout 10%, and in many cases, less than about 5%, while havingunoccupied droplets of less than about 50%, less than about 40%, lessthan about 30%, less than about 20%, less than about 10%, less thanabout 5%, or less.

The flow of the first fluid may be such that the droplets contain asingle particle, e.g., bead. In certain embodiments, the yield ofdroplets containing a single particle is at least 80%, e.g., at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99%.

As will be appreciated, the above-described occupancy rates are alsoapplicable to droplets that include both biological particles (e.g.,cells) and beads. The occupied droplets (e.g., at least about 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the occupied droplets)can include both a bead and a biological particle. Particles, e.g.,beads, within a channel (e.g., a particle channel) may flow at asubstantially regular flow profile (e.g., at a regular flow rate) toprovide a droplet, when formed, with a single particle (e.g., bead) anda single cell or other biological particle. Such regular flow profilesmay permit the droplets to have a dual occupancy (e.g., droplets havingat least one bead and at least one cell or biological particle) greaterthan 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or99%. In some embodiments, the droplets have a 1:1 dual occupancy (i.e.,droplets having exactly one particle (e.g., bead) and exactly one cellor biological particle) greater than 5%, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97% 98%, or 99%. Such regular flow profiles anddevices that may be used to provide such regular flow profiles areprovided, for example, in U.S. Patent Publication No. 2015/0292988,which is entirely incorporated herein by reference.

In some cases, additional particles may be used to deliver additionalreagents to a droplet. In such cases, it may be advantageous tointroduce different particles (e.g., beads) into a common channel (e.g.,proximal to or upstream from a droplet formation region) or dropletformation intersection from different bead sources (e.g., containingdifferent associated reagents) through different channel inlets intosuch common channel or droplet formation region. In such cases, the flowand/or frequency of each of the different particle, e.g., bead, sourcesinto the channel or fluidic connections may be controlled to provide forthe desired ratio of particles, e.g., beads, from each source, whileoptionally ensuring the desired pairing or combination of suchparticles, e.g., beads, are formed into a droplet with the desirednumber of biological particles.

The droplets described herein may comprise small volumes, for example,less than about 10 microliters (μL), 5 μL, 1 μL, 900 picoliters (pL),800 pL, 700 pL, 600 pL, 500 pL, 400 pL, 300 pL, 200 pL, 100 pL, 50 pL,20 pL, 10 pL, 1 pL, 500 nanoliters (nL), 100 nL, 50 nL, or less. Forexample, the droplets may have overall volumes that are less than about1000 pL, 900 pL, 800 pL, 700 pL, 600 pL, 500 pL, 400pL, 300 pL, 200 pL,100pL, 50 pL, 20 pL, 10 pL, 1 pL, or less. Where the droplets furthercomprise particles (e.g., beads or microcapsules), it will beappreciated that the sample liquid volume within the droplets may beless than about 90% of the above described volumes, less than about 80%,less than about 70%, less than about 60%, less than about 50%, less thanabout 40%, less than about 30%, less than about 20%, or less than about10% the above described volumes (e.g., of a partitioning liquid), e.g.,from 1% to 99%, from 5% to 95%, from 10% to 90%, from 20% to 80%, from30% to 70%, or from 40% to 60%, e.g., from 1% to 5%, 5% to 10%, 10% to15%, 15% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to45%, 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70%, 70% to75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100% ofthe above described volumes.

Any suitable number of droplets can be generated. For example, in amethod described herein, a plurality of droplets may be generated thatcomprises at least about 1,000 droplets, at least about 5,000 droplets,at least about 10,000 droplets, at least about 50,000 droplets, at leastabout 100,000 droplets, at least about 500,000 droplets, at least about1,000,000 droplets, at least about 5,000,000 droplets at least about10,000,000 droplets, at least about 50,000,000 droplets, at least about100,000,000 droplets, at least about 500,000,000 droplets, at leastabout 1,000,000,000 droplets, or more. Moreover, the plurality ofdroplets may comprise both unoccupied droplets (e.g., empty droplets)and occupied droplets.

The fluid to be dispersed into droplets may be transported from areservoir to the droplet formation region. Alternatively, the fluid tobe dispersed into droplets is formed in situ by combining two or morefluids in the device. For example, the fluid to be dispersed may beformed by combining one fluid containing one or more reagents with oneor more other fluids containing one or more reagents. In theseembodiments, the mixing of the fluid streams may result in a chemicalreaction. For example, when a particle is employed, a fluid havingreagents that disintegrates the particle may be combined with theparticle, e.g., immediately upstream of the droplet generating region.In these embodiments, the particles may be cells, which can be combinedwith lysing reagents, such as surfactants. When particles, e.g., beads,are employed, the particles, e.g., beads, may be dissolved or chemicallydegraded, e.g., by a change in pH (acid or base), redox potential (e.g.,addition of an oxidizing or reducing agent), enzymatic activity, changein salt or ion concentration, or other mechanism.

The first fluid is transported through the first channel at a flow ratesufficient to produce droplets in the droplet formation region. Fasterflow rates of the first fluid generally increase the rate of dropletproduction; however, at a high enough rate, the first fluid will form ajet, which may not break up into droplets. Typically, the flow rate ofthe first fluid though the first channel may be between about 0.01μL/min to about 100 μL/min, e.g., 0.1 to 50 μL/min, 0.1 to 10 A/min, or1 to 5 μL/min. In some instances, the flow rate of the first liquid maybe between about 0.04 μL/min and about 40 μL/min. In some instances, theflow rate of the first liquid may be between about 0.01 μL/min and about100 μL/min.

Alternatively, the flow rate of the first liquid may be less than about0.01 μL/min. Alternatively, the flow rate of the first liquid may begreater than about 40 μL/min, e.g., 45 μL/min, 50 μL/min, 55 μL/min, 60μL/min, 65 μL/min, 70 μL/min, 75 μL/min, 80 μL/min, 85 μL/min, 90μL/min, 95 μL/min, 100 μL/min, 110 μL/min, 120 μL/min, 130 μL/min, 140μL/min, 150 μL/min, or greater. At lower flow rates, such as flow ratesof about less than or equal to 10 A/min, the droplet radius may not bedependent on the flow rate of first liquid. Alternatively or inaddition, for any of the abovementioned flow rates, the droplet radiusmay be independent of the flow rate of the first liquid.

The typical droplet formation rate for a single channel in a device ofthe invention is between 0.1 Hz to 10,000 Hz, e.g., 1 to 1000 Hz or 1 to500 Hz. The use of multiple first channels can increase the rate ofdroplet formation by increasing the number of locations of formation.

As discussed above, droplet formation may occur in the absence ofexternally driven movement of the continuous phase. In such embodiments,the continuous phase flows in response to displacement by the advancingstream of the first fluid or other forces. Channels may be present inthe droplet formation region, e.g., including a shelf region, to allowmore rapid transport of the continuous phase around the first fluid.This increase in transport of the continuous phase can increase the rateof droplet formation.

Alternatively, the continuous phase may be actively transported. Forexample, the continuous phase may be actively transported into thedroplet formation region, e.g., including a shelf region, to increasethe rate of droplet formation; continuous phase may be activelytransported to form a sheath flow around the first fluid as it exits thedistal end; or the continuous phase may be actively transported to movedroplets away from the point of formation.

Additional factors that affect the rate of droplet formation include theviscosity of the first fluid and of the continuous phase, whereincreasing the viscosity of either fluid reduces the rate of dropletformation. In certain embodiments, the viscosity of the first fluidand/or continuous is between 0.5 cP to 10 cP. Furthermore, lowerinterfacial tension results in slower droplet formation. In certainembodiments, the interfacial tension is between 0.1 and 100 mN/m, e.g.,1 to 100 mN/m or 2 mN/m to 60 mN/m. The depth of the shelf region canalso be used to control the rate of droplet formation, with a shallowerdepth resulting in a faster rate of formation.

The methods may be used to produce droplets in range of 1 μm to 500 μmin diameter, e.g., 1 to 250 μm, 5 to 200 μm, 5 to 150 μm, or 12 to 125μm. Factors that affect the size of the droplets include the rate offormation, the cross-sectional dimension of the distal end of the firstchannel, the depth of the shelf, and fluid properties and dynamiceffects, such as the interfacial tension, viscosity, and flow rate.

The first liquid may be aqueous, and the second liquid may be an oil (orvice versa). Examples of oils include perfluorinated oils, mineral oil,and silicone oils. For example, a fluorinated oil may include afluorosurfactant for stabilizing the resulting droplets, for example,inhibiting subsequent coalescence of the resulting droplets. Examples ofparticularly useful liquids and fluorosurfactants are described, forexample, in U.S. Pat. No. 9,012,390, which is entirely incorporatedherein by reference for all purposes. Specific examples includehydrofluoroethers, such as HFE 7500, 7300, 7200, or 7100. Suitableliquids are those described in US 2015/0224466 and U.S. 62/522,292, theliquids of which are hereby incorporated by reference. In some cases,liquids include additional components such as a particle, e.g., a cellor a gel bead. As discussed above, the first fluid or continuous phasemay include reagents for carrying out various reactions, such as nucleicacid amplification, lysis, or bead dissolution. The first liquid orcontinuous phase may include additional components that stabilize orotherwise affect the droplets or a component inside the droplet. Suchadditional components include surfactants, antioxidants, preservatives,buffering agents, antibiotic agents, salts, chaotropic agents, enzymes,nanoparticles, and sugars.

Devices, systems, compositions, and methods of the present disclosuremay be used for various applications, such as, for example, processing asingle analyte (e.g., bioanalytes, e.g., RNA, DNA, or protein) ormultiple analytes (e.g., bioanalytes, e.g., DNA and RNA, DNA andprotein, RNA and protein, or RNA, DNA and protein) from a single cell.For example, a biological particle (e.g., a cell or virus) can be formedin a droplet, and one or more analytes (e.g., bioanalytes) from thebiological particle (e.g., cell) can be modified or detected (e.g.,bound, labeled, or otherwise modified by an analyte detection moiety)for subsequent processing. The multiple analytes may be from the singlecell. This process may enable, for example, proteomic, transcriptomic,and/or genomic analysis of the cell or population thereof (e.g.,simultaneous proteomic, transcriptomic, and/or genomic analysis of thecell or population thereof).

Methods of modifying analytes include providing a plurality of particles(e.g., beads) in a liquid carrier (e.g., an aqueous carrier); providinga sample containing an analyte (e.g., as part of a cell, or component orproduct thereof) in a sample liquid; and using the device to combine theliquids and form an analyte detection droplet containing one or moreparticles and one or more analytes (e.g., as part of one or more cells,or components or products thereof). Such sequestration of one or moreparticles with analyte (e.g., bioanalyte associated with a cell) in adroplet enables labeling of discrete portions of large, heterologoussamples (e.g., single cells within a heterologous population). Oncelabeled or otherwise modified, droplets can be combined (e.g., bybreaking an emulsion), and the resulting liquid can be analyzed todetermine a variety of properties associated with each of numeroussingle cells.

In particular embodiments, the invention features methods of producinganalyte detection droplets using a device having a particle channel anda sample channel that intersect proximal to a droplet formation region.Particles having an analyte-detection moiety in a liquid carrier flowproximal-to-distal (e.g., towards the droplet formation region) throughthe particle channel and a sample liquid containing an analyte flowsproximal-to-distal (e.g., towards the droplet formation region) throughthe sample channel until the two liquids meet and combine at theintersection of the sample channel and the particle channel, upstream(and/or proximal to) the droplet formation region. The combination ofthe liquid carrier with the sample liquid results in an analytedetection liquid. In some embodiments, the two liquids are miscible(e.g., they both contain solutes in water or aqueous buffer). Thecombination of the two liquids can occur at a controlled relative rate,such that the analyte detection liquid has a desired volumetric ratio ofparticle liquid to sample liquid, a desired numeric ratio of particlesto cells, or a combination thereof (e.g., one particle per cell per 50pL). As the analyte detection liquid flows through the droplet formationregion into a partitioning liquid (e.g., a liquid which is immisciblewith the analyte detection liquid, such as an oil), analyte detectiondroplets form. These analyte detection droplets may continue to flowthrough one or more channels. Alternatively or in addition, the analytedetection droplets may accumulate (e.g., as a substantially stationarypopulation) in a droplet collection region. In some cases, theaccumulation of a population of droplets may occur by a gentle flow of afluid within the droplet collection region, e.g., to move the formeddroplets out of the path of the nascent droplets.

Devices useful for analyte detection may feature any combination ofelements described herein. For example, various droplet formationregions can be employed in the design of a device for analyte detection.In some embodiments, analyte detection droplets are formed at a dropletformation region having a shelf region, where the analyte detectionliquid expands in at least one dimension as it passes through thedroplet formation region. Any shelf region described herein can beuseful in the methods of analyte detection droplet formation providedherein. Additionally or alternatively, the droplet formation region mayhave a step at or distal to an inlet of the droplet formation region(e.g., within the droplet formation region or distal to the dropletformation region). In some embodiments, analyte detection droplets areformed without externally driven flow of a continuous phase (e.g., byone or more crossing flows of liquid at the droplet formation region).Alternatively, analyte detection droplets are formed in the presence ofan externally driven flow of a continuous phase.

A device useful for droplet formation, e.g., analyte detection, mayfeature multiple droplet formation regions (e.g., in or out of (e.g., asindependent, parallel circuits) fluid communication with one another.For example, such a device may have 2-100, 3-50, 4-40, 5-30, 6-24, 8-18,or 9-12, e.g., 2-6, 6-12, 12-18, 18-24, 24-36, 36-48, or 48-96, e.g., 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, or more droplet formation regionsconfigured to produce analyte detection droplets).

Source reservoirs can store liquids prior to and during dropletformation. In some embodiments, a device useful in analyte detectiondroplet formation includes one or more particle reservoirs connectedproximally to one or more particle channels. Particle suspensions can bestored in particle reservoirs prior to analyte detection dropletformation. Particle reservoirs can be configured to store particlescontaining an analyte detection moiety. For example, particle reservoirscan include, e.g., a coating to prevent adsorption or binding (e.g.,specific or non-specific binding) of particles or analyte-detectionmoieties. Additionally or alternatively, particle reservoirs can beconfigured to minimize degradation of analyte detection moieties (e.g.,by containing nuclease, e.g., DNAse or RNAse) or the particle matrixitself, accordingly.

Additionally or alternatively, a device includes one or more samplereservoirs connected proximally to one or more sample channels. Samplescontaining cells and/or other reagents useful in analyte detectionand/or droplet formation can be stored in sample reservoirs prior toanalyte detection droplet formation. Sample reservoirs can be configuredto reduce degradation of sample components, e.g., by including nuclease(e.g., DNAse or RNAse).

Methods of the invention include administering a sample and/or particlesto the device, for example, (a) by pipetting a sample liquid, or acomponent or concentrate thereof, into a sample reservoir and/or (b) bypipetting a liquid carrier (e.g., an aqueous carrier) and/or particlesinto a particle reservoir. In some embodiments, the method involvesfirst pipetting the liquid carrier (e.g., an aqueous carrier) and/orparticles into the particle reservoir prior to pipetting the sampleliquid, or a component or concentrate thereof, into the samplereservoir.

The sample reservoir and/or particle reservoir may be incubated inconditions suitable to preserve or promote activity of their contentsuntil the initiation or commencement of droplet formation.

Formation of bioanalyte detection droplets, as provided herein, can beused for various applications. In particular, by forming bioanalytedetection droplets using the methods, devices, systems, and kits herein,a user can perform standard downstream processing methods to barcodeheterogeneous populations of cells or perform single-cell nucleic acidsequencing.

In methods of barcoding a population of cells, an aqueous sample havinga population of cells is combined with bioanalyte detection particleshaving a nucleic acid primer sequence and a barcode in an aqueouscarrier at an intersection of the sample channel and the particlechannel to form a reaction liquid.

Upon passing through the droplet formation region, the reaction liquidmeets a partitioning liquid (e.g., a partitioning oil) underdroplet-forming conditions to form a plurality of reaction droplets,each reaction droplet having one or more of the particles and one ormore cells in the reaction liquid. The reaction droplets are incubatedunder conditions sufficient to allow for barcoding of the nucleic acidof the cells in the reaction droplets. In some embodiments, theconditions sufficient for barcoding are thermally optimized for nucleicacid replication, transcription, and/or amplification. For example,reaction droplets can be incubated at temperatures configured to enablereverse transcription of RNA produced by a cell in a droplet into DNA,using reverse transcriptase. Additionally or alternatively, reactiondroplets may be cycled through a series of temperatures to promoteamplification, e.g., as in a polymerase chain reaction (PCR).Accordingly, in some embodiments, one or more nucleotide amplificationreagents (e.g., PCR reagents) are included in the reaction droplets(e.g., primers, nucleotides, and/or polymerase). Any one or morereagents for nucleic acid replication, transcription, and/oramplification can be provided to the reaction droplet by the aqueoussample, the liquid carrier, or both. In some embodiments, one or more ofthe reagents for nucleic acid replication, transcription, and/oramplification are in the aqueous sample.

Also provided herein are methods of single-cell nucleic acid sequencing,in which a heterologous population of cells can be characterized bytheir individual gene expression, e.g., relative to other cells of thepopulation. Methods of barcoding cells discussed above and known in theart can be part of the methods of single-cell nucleic acid sequencingprovided herein. After barcoding, nucleic acid transcripts that havebeen barcoded are sequenced, and sequences can be processed, analyzed,and stored according to known methods. In some embodiments, thesemethods enable the generation of a genome library containing geneexpression data for any single cell within a heterologous population.

Alternatively, the ability to sequester a single cell in a reactiondroplet provided by methods herein enables bioanalyte detection forapplications beyond genome characterization. For example, a reactiondroplet containing a single cell and variety of analyte detectionmoieties capable of binding different proteins can allow a single cellto be detectably labeled to provide relative protein expression data. Insome embodiments, analyte detection moieties are antigen-bindingmolecules (e.g., antibodies or fragments thereof), wherein each antibodyclone is detectably labeled (e.g., with a fluorescent marker having adistinct emission wavelength). Binding of antibodies to proteins canoccur within the reaction droplet, and cells can be subsequentlyanalyzed for bound antibodies according to known methods to generate alibrary of protein expression. Other methods known in the art can beemployed to characterize cells within heterologous populations afterdetecting analytes using the methods provided herein. In one example,following the formation or droplets, subsequent operations that can beperformed can include formation of amplification products, purification(e.g., via solid phase reversible immobilization (SPRI)), furtherprocessing (e.g., shearing, ligation of functional sequences, andsubsequent amplification (e.g., via PCR)). These operations may occur inbulk (e.g., outside the droplet). An exemplary use for droplets formedusing methods of the invention is in performing nucleic acidamplification, e.g., polymerase chain reaction (PCR), where the reagentsnecessary to carry out the amplification are contained within the firstfluid. In the case where a droplet is a droplet in an emulsion, theemulsion can be broken and the contents of the droplet pooled foradditional operations. Additional reagents that may be included in adroplet along with the barcode bearing bead may include oligonucleotidesto block ribosomal RNA (rRNA) and nucleases to digest genomic DNA fromcells. Alternatively, rRNA removal agents may be applied duringadditional processing operations. The configuration of the constructsgenerated by such a method can help minimize (or avoid) sequencing ofpoly-T sequence during sequencing and/or sequence the 5′ end of apolynucleotide sequence. The amplification products, for example firstamplification products and/or second amplification products, may besubject to sequencing for sequence analysis. In some cases,amplification may be performed using the Partial Hairpin Amplificationfor Sequencing (PHASE) method.

Methods of Device Manufacture

The microfluidic devices of the present disclosure may be fabricated inany of a variety of conventional ways. For example, in some cases thedevices comprise layered structures, where a first layer includes aplanar surface into which is disposed a series of channels or groovesthat correspond to the channel network in the finished device. A secondlayer includes a planar surface on one side, and a series of reservoirsdefined on the opposing surface, where the reservoirs communicate aspassages through to the planar layer, such that when the planar surfaceof the second layer is mated with the planar surface of the first layer,the reservoirs defined in the second layer are positioned in liquidcommunication with the termini of the channels on the first layer.Alternatively, both the reservoirs and the connected channels may befabricated into a single part, where the reservoirs are provided upon afirst surface of the structure, with the apertures of the reservoirsextending through to the opposing surface of the structure. The channelnetwork is fabricated as a series of grooves and features in this secondsurface. A thin laminating layer is then provided over the secondsurface to seal, and provide the final wall of the channel network, andthe bottom surface of the reservoirs.

These layered structures may be fabricated in whole or in part frompolymeric materials, such as polyethylene or polyethylene derivatives,such as cyclic olefin copolymers (COC), polymethylmethacrylate (PMMA),polydimethylsiloxane (PDMS), polycarbonate, polystyrene, polypropylene,polyvinyl chloride, polytetrafluoroethylene, polyoxymethylene, polyetherether ketone, polycarbonate, polystyrene, or the like, or they may befabricated in whole or in part from inorganic materials, such assilicon, or other silica based materials, e.g., glass, quartz, fusedsilica, borosilicate glass, metals, ceramics, and combinations thereof.Polymeric device components may be fabricated using any of a number ofprocesses including soft lithography, embossing techniques,micromachining, e.g., laser machining, or in some aspects injectionmolding of the layer components that include the defined channels aswell as other structures, e.g., reservoirs, integrated functionalcomponents, etc. In some aspects, the structure comprising thereservoirs and channels may be fabricated using, e.g., injection moldingtechniques to produce polymeric structures. In such cases, a laminatinglayer may be adhered to the molded structured part through readilyavailable methods, including thermal lamination, solvent basedlamination, sonic welding, or the like.

As will be appreciated, structures comprised of inorganic materials alsomay be fabricated using known techniques. For example, channels andother structures may be micro-machined into surfaces or etched into thesurfaces using standard photolithographic techniques. In some aspects,the microfluidic devices or components thereof may be fabricated usingthree-dimensional printing techniques to fabricate the channel or otherstructures of the devices and/or their discrete components.

Methods for Surface Modifications

The invention features methods for producing a microfluidic device thathas a surface modification, e.g., a surface with a modified watercontact angle. The methods may be employed to modify the surface of adevice such that a liquid can “wet” the surface by altering the contactangle the liquid makes with the surface. An exemplary use of the methodsof the invention is in creating a device having differentially coatedsurfaces to optimize droplet formation.

Devices to be modified with surface coating agents may be primed, e.g.,pre-treated, before coating processes occur. In one embodiment, thedevice has a channel that is in fluid communication with a dropletformation region. In particular, the droplet formation region isconfigured to allow a liquid exiting the channel to expand in at leastone dimension. A surface of the droplet formation region is contacted byat least one reagent that has an affinity for the primed surface toproduce a surface having a first water contact angle of greater thanabout 90°, e.g., a hydrophobic or fluorophillic surface. In certainembodiments, the first contact angle is greater than the water contactangle of the primed surface. In other embodiments, the first contactangle is greater than the water contact angle of the channel surface.Thus, the method allows for the differential coating of surfaces withinthe microfluidic device.

A surface may be primed by depositing a metal oxide onto it. Examplemetal oxides useful for priming surfaces include, but are not limitedto, Al₂O₃, TiO₂, SiO₂, or a combination thereof. Other metal oxidesuseful for surface modifications are known in the art. The metal oxidecan be applied to the surface by standard deposition techniques,including, but not limited to, atomic layer deposition (ALD), physicalvapor deposition (PVD), e.g., sputtering, chemical vapor deposition(CVD), or laser deposition. Other deposition techniques for coatingsurfaces, e.g., liquid-based deposition, are known in the art. Forexample, an atomic layer of A1203 can be prepared on a surface bydepositing trimethylaluminum (TMA) and water.

In some cases, the coating agent may create a surface that has a watercontact angle greater than 90°, e.g., hydrophobic or fluorophillic, ormay create a surface with a water contact angle of less than 90°, e.g.,hydrophilic. For example, a fluorophillic surface may be created byflowing fluorosilane (e.g., H3FSi) through a primed device surface,e.g., a surface coated in a metal oxide. The priming of the surfaces ofthe device enhances the adhesion of the coating agents to the surface byproviding appropriate surface functional groups. In some cases, thecoating agent used to coat the primed surface may be a liquid reagent.For example, when a liquid coating agent is used to coat a surface, thecoating agent may be directly introduced to the droplet formation regionby a feed channel in fluid communication with the droplet formationregion. In order to keep the coating agent localized to the dropletformation region, e.g., prevent ingress of the coating agent to anotherportion of the device, e.g., the channel, the portion of the device thatis not to be coated can be substantially blocked by a substance thatdoes not allow the coating agent to pass. For example, in order toprevent ingress of a liquid coating agent into the channel, the channelmay be filled with a blocking liquid that is substantially immisciblewith the coating agent. The blocking liquid may be actively transportedthrough the portion of the device not to be coated, or the blockingliquid may be stationary. Alternatively, the channel may be filled witha pressurized gas such that the pressure prevents ingress of the coatingagent into the channel. The coating agent may also be applied to theregions of interest external to the main device. For example, the devicemay incorporate an additional reservoir and at least one feed channelthat connects to the region of interest such that no coating agent ispassed through the device.

EXAMPLES Example 1

FIG. 1 shows an example of a microfluidic device for the controlledinclusion of particles, e.g., beads, into discrete droplets. A device100 can include a channel 102 communicating at a fluidic connection 106(or intersection) with a reservoir 104. The reservoir 104 can be achamber. Any reference to “reservoir,” as used herein, can also refer toa “chamber.” In operation, an aqueous liquid 108 that includes suspendedbeads 112 may be transported along the channel 102 into the fluidicconnection 106 to meet a second liquid 110 that is immiscible with theaqueous liquid 108 in the reservoir 104 to create droplets 116, 118 ofthe aqueous liquid 108 flowing into the reservoir 104. At the fluidicconnection 106 where the aqueous liquid 108 and the second liquid 110meet, droplets can form based on factors such as the hydrodynamic forcesat the fluidic connection 106, flow rates of the two liquids 108, 110,liquid properties, and certain geometric parameters (e.g., w, h₀, α,etc.) of the device 100. A plurality of droplets can be collected in thereservoir 104 by continuously injecting the aqueous liquid 108 from thechannel 102 through the fluidic connection 106.

In some instances, the second liquid 110 may not be subjected to and/ordirected to any flow in or out of the reservoir 104. For example, thesecond liquid 110 may be substantially stationary in the reservoir 104.In some instances, the second liquid 110 may be subjected to flow withinthe reservoir 104, but not in or out of the reservoir 104, such as viaapplication of pressure to the reservoir 104 and/or as affected by theincoming flow of the aqueous liquid 108 at the fluidic connection 106.Alternatively, the second liquid 110 may be subjected and/or directed toflow in or out of the reservoir 104. For example, the reservoir 104 canbe a channel directing the second liquid 110 from upstream todownstream, transporting the generated droplets. Alternatively or inaddition, the second liquid 110 in reservoir 104 may be used to sweepformed droplets away from the path of the nascent droplets.

While FIG. 1 illustrates the reservoir 104 having a substantially linearinclination (e.g., creating the expansion angle, α) relative to thechannel 102, the inclination may be non-linear. The expansion angle maybe an angle between the immediate tangent of a sloping inclination andthe channel 102. In an example, the reservoir 104 may have a dome-like(e.g., hemispherical) shape. The reservoir 104 may have any other shape.

Example b 2

FIG. 2 shows an example of a microfluidic device for increased dropletformation throughput. A device 200 can comprise a plurality of channels202 and a reservoir 204. Each of the plurality of channels 202 may be influid communication with the reservoir 204. The device 200 can comprisea plurality of fluidic connections 206 between the plurality of channels202 and the reservoir 204. Each fluidic connection can be a point ofdroplet formation. The channel 102 from the device 100 in FIG. 1 and anydescription to the components thereof may correspond to a given channelof the plurality of channels 202 in device 200 and any description tothe corresponding components thereof. The reservoir 104 from the device100 and any description to the components thereof may correspond to thereservoir 204 from the device 200 and any description to thecorresponding components thereof.

Each channel of the plurality of channels 202 may comprise an aqueousliquid 208 that includes suspended particles, e.g., beads, 212. Thereservoir 204 may comprise a second liquid 210 that is immiscible withthe aqueous liquid 208. In some instances, the second liquid 210 may notbe subjected to and/or directed to any flow in or out of the reservoir204. For example, the second liquid 210 may be substantially stationaryin the reservoir 204. Alternatively or in addition, the formed dropletscan be moved out of the path of nascent droplets using a gentle flow ofthe second liquid 210 in the reservoir 204. In some instances, thesecond liquid 210 may be subjected to flow within the reservoir 204, butnot in or out of the reservoir 204, such as via application of pressureto the reservoir 204 and/or as affected by the incoming flow of theaqueous liquid 208 at the fluidic connections. Alternatively, the secondliquid 210 may be subjected and/or directed to flow in or out of thereservoir 204. For example, the reservoir 204 can be a channel directingthe second liquid 210 from upstream to downstream, transporting thegenerated droplets. Alternatively or in addition, the second liquid 210in reservoir 204 may be used to sweep formed droplets away from the pathof the nascent droplets.

In operation, the aqueous liquid 208 that includes suspended particles,e.g., beads, 212 may be transported along the plurality of channels 202into the plurality of fluidic connections 206 to meet the second liquid210 in the reservoir 204 to create droplets 216, 218. A droplet may formfrom each channel at each corresponding fluidic connection with thereservoir 204. At the fluidic connection where the aqueous liquid 208and the second liquid 210 meet, droplets can form based on factors suchas the hydrodynamic forces at the fluidic connection, flow rates of thetwo liquids 208, 210, liquid properties, and certain geometricparameters (e.g., w, h₀, α, etc.) of the device 200, as describedelsewhere herein. A plurality of droplets can be collected in thereservoir 204 by continuously injecting the aqueous liquid 208 from theplurality of channels 202 through the plurality of fluidic connections206. The geometric parameters, w, h₀, and α, may or may not be uniformfor each of the channels in the plurality of channels 202. For example,each channel may have the same or different widths at or near itsrespective fluidic connection with the reservoir 204. For example, eachchannel may have the same or different height at or near its respectivefluidic connection with the reservoir 204. In another example, thereservoir 204 may have the same or different expansion angle at thedifferent fluidic connections with the plurality of channels 202. Whenthe geometric parameters are uniform, beneficially, droplet size mayalso be controlled to be uniform even with the increased throughput. Insome instances, when it is desirable to have a different distribution ofdroplet sizes, the geometric parameters for the plurality of channels202 may be varied accordingly.

Example 3

FIG. 3 shows another example of a microfluidic device for increaseddroplet formation throughput. A microfluidic device 300 can comprise aplurality of channels 302 arranged generally circularly around theperimeter of a reservoir 304. Each of the plurality of channels 302 maybe in liquid communication with the reservoir 304. The device 300 cancomprise a plurality of fluidic connections 306 between the plurality ofchannels 302 and the reservoir 304. Each fluidic connection can be apoint of droplet formation. The channel 102 from the device 100 in FIG.1 and any description to the components thereof may correspond to agiven channel of the plurality of channels 302 in device 300 and anydescription to the corresponding components thereof. The reservoir 104from the device 100 and any description to the components thereof maycorrespond to the reservoir 304 from the device 300 and any descriptionto the corresponding components thereof.

Each channel of the plurality of channels 302 may comprise an aqueousliquid 308 that includes suspended particles, e.g., beads, 312. Thereservoir 304 may comprise a second liquid 310 that is immiscible withthe aqueous liquid 308. In some instances, the second liquid 310 may notbe subjected to and/or directed to any flow in or out of the reservoir304. For example, the second liquid 310 may be substantially stationaryin the reservoir 304. In some instances, the second liquid 310 may besubjected to flow within the reservoir 304, but not in or out of thereservoir 304, such as via application of pressure to the reservoir 304and/or as affected by the incoming flow of the aqueous liquid 308 at thefluidic connections. Alternatively, the second liquid 310 may besubjected and/or directed to flow in or out of the reservoir 304. Forexample, the reservoir 304 can be a channel directing the second liquid310 from upstream to downstream, transporting the generated droplets.Alternatively or in addition, the second liquid 310 in reservoir 304 maybe used to sweep formed droplets away from the path of the nascentdroplets.

In operation, the aqueous liquid 308 that includes suspended particles,e.g., beads, 312 may be transported along the plurality of channels 302into the plurality of fluidic connections 306 to meet the second liquid310 in the reservoir 304 to create a plurality of droplets 316. Adroplet may form from each channel at each corresponding fluidicconnection with the reservoir 304. At the fluidic connection where theaqueous liquid 308 and the second liquid 310 meet, droplets can formbased on factors such as the hydrodynamic forces at the fluidicconnection, flow rates of the two liquids 308, 310, liquid properties,and certain geometric parameters (e.g., widths and heights of thechannels 302, expansion angle of the reservoir 304, etc.) of the channel300, as described elsewhere herein. A plurality of droplets can becollected in the reservoir 304 by continuously injecting the aqueousliquid 308 from the plurality of channels 302 through the plurality offluidic connections 306.

Example 4

FIG. 4 shows another example of a microfluidic device for theintroduction of beads into discrete droplets. A device 400 can include afirst channel 402, a second channel 404, a third channel 404, a fourthchannel 406, and a reservoir 410. The first channel 402, second channel404, third channel 404, and fourth channel 406 can communicate at afirst intersection 418. The fourth channel 406 and the reservoir 410 cancommunicate at a fluidic connection 422. In some instances, the fourthchannel 406 and components thereof can correspond to the channel 102 inthe device 100 in FIG. 1 and components thereof. In some instances, thereservoir 410 and components thereof can correspond to the reservoir 104in the device 100 and components thereof.

In operation, an aqueous liquid 412 that includes suspended particles,e.g., beads, 416 may be transported along the first channel 402 into theintersection 418 at a first frequency to meet another source of theaqueous liquid 412 flowing along the second channel 404 and the thirdchannel 406 towards the intersection 418 at a second frequency. In someinstances, the aqueous liquid 412 in the second channel 404 and thethird channel 406 may comprise one or more reagents. At theintersection, the combined aqueous liquid 412 carrying the suspendedparticles, e.g., beads, 416 (and/or the reagents) can be directed intothe fourth channel 408. In some instances, a cross-section width ordiameter of the fourth channel 408 can be chosen to be less than across-section width or diameter of the particles, e.g., beads, 416. Insuch cases, the particles, e.g., beads, 416 can deform and travel alongthe fourth channel 408 as deformed particles, e.g., beads, 420 towardsthe fluidic connection 422. At the fluidic connection 422, the aqueousliquid 412 can meet a second liquid 414 that is immiscible with theaqueous liquid 412 in the reservoir 410 to create droplets 420 of theaqueous liquid 412 flowing into the reservoir 410. Upon leaving thefourth channel 408, the deformed particles, e.g., beads, 420 may revertto their original shape in the droplets 420. At the fluidic connection422 where the aqueous liquid 412 and the second liquid 414 meet,droplets can form based on factors such as the hydrodynamic forces atthe fluidic connection 422, flow rates of the two liquids 412, 414,liquid properties, and certain geometric parameters (e.g., w, h₀, α,etc.) of the channel 400, as described elsewhere herein. A plurality ofdroplets can be collected in the reservoir 410 by continuously injectingthe aqueous liquid 412 from the fourth channel 408 through the fluidicconnection 422.

A discrete droplet generated may include a particle, e.g., a bead,(e.g., as in droplets 420). Alternatively, a discrete droplet generatedmay include more than one particle, e.g., bead. Alternatively, adiscrete droplet generated may not include any particles, e.g., beads.In some instances, a discrete droplet generated may contain one or morebiological particles, e.g., cells (not shown in FIG. 4).

In some instances, the second liquid 414 may not be subjected to and/ordirected to any flow in or out of the reservoir 410. For example, thesecond liquid 414 may be substantially stationary in the reservoir 410.In some instances, the second liquid 414 may be subjected to flow withinthe reservoir 410, but not in or out of the reservoir 410, such as viaapplication of pressure to the reservoir 410 and/or as affected by theincoming flow of the aqueous liquid 412 at the fluidic connection 422.In some instances, the second liquid 414 may be gently stirred in thereservoir 410. Alternatively, the second liquid 414 may be subjectedand/or directed to flow in or out of the reservoir 410. For example, thereservoir 410 can be a channel directing the second liquid 414 fromupstream to downstream, transporting the generated droplets.Alternatively or in addition, the second liquid 414 in reservoir 410 maybe used to sweep formed droplets away from the path of the nascentdroplets.

Example 5

FIG. 5A shows a cross-section view of another example of a microfluidicdevice with a geometric feature for droplet formation. A device 500 caninclude a channel 502 communicating at a fluidic connection 506 (orintersection) with a reservoir 504. In some instances, the device 500and one or more of its components can correspond to the device 100 andone or more of its components. FIG. 7B shows a perspective view of thedevice 500 of FIG. 7A.

An aqueous liquid 512 comprising a plurality of particles 516 may betransported along the channel 502 into the fluidic connection 506 tomeet a second liquid 514 (e.g., oil, etc.) that is immiscible with theaqueous liquid 512 in the reservoir 504 to create droplets 520 of theaqueous liquid 512 flowing into the reservoir 504. At the fluidicconnection 506 where the aqueous liquid 512 and the second liquid 514meet, droplets can form based on factors such as the hydrodynamic forcesat the fluidic connection 506, relative flow rates of the two liquids512, 514, liquid properties, and certain geometric parameters (e.g., h,etc.) of the device 500. A plurality of droplets can be collected in thereservoir 504 by continuously injecting the aqueous liquid 512 from thechannel 502 at the fluidic connection 506.

While FIGS. 5A and 5B illustrate the height difference, Δh, being abruptat the fluidic connection 506 (e.g., a step increase), the heightdifference may increase gradually (e.g., from about 0 μm to a maximumheight difference). Alternatively, the height difference may decreasegradually (e.g., taper) from a maximum height difference. A gradualincrease or decrease in height difference, as used herein, may refer toa continuous incremental increase or decrease in height difference,wherein an angle between any one differential segment of a heightprofile and an immediately adjacent differential segment of the heightprofile is greater than 90°. For example, at the fluidic connection 506,a bottom wall of the channel and a bottom wall of the reservoir can meetat an angle greater than 90°. Alternatively or in addition, a top wall(e.g., ceiling) of the channel and a top wall (e.g., ceiling) of thereservoir can meet an angle greater than 90°. A gradual increase ordecrease may be linear or non-linear (e.g., exponential, sinusoidal,etc.). Alternatively or in addition, the height difference may variablyincrease and/or decrease linearly or non-linearly.

Example 6

FIGS. 6A and 6B show a cross-section view and a top view, respectively,of another example of a microfluidic device with a geometric feature fordroplet formation. A device 600 can include a channel 602 communicatingat a fluidic connection 606 (or intersection) with a reservoir 604. Insome instances, the device 600 and one or more of its components cancorrespond to the channel 500 and one or more of its components.

An aqueous liquid 612 comprising a plurality of particles 616 may betransported along the channel 602 into the fluidic connection 606 tomeet a second liquid 614 (e.g., oil, etc.) that is immiscible with theaqueous liquid 612 in the reservoir 604 to create droplets 620 of theaqueous liquid 612 flowing into the reservoir 604. At the fluidicconnection 606 where the aqueous liquid 612 and the second liquid 614meet, droplets can form based on factors such as the hydrodynamic forcesat the fluidic connection 606, relative flow rates of the two liquids612, 614, liquid properties, and certain geometric parameters (e.g., dh,ledge, etc.) of the channel 602. A plurality of droplets can becollected in the reservoir 604 by continuously injecting the aqueousliquid 612 from the channel 602 at the fluidic connection 606.

The aqueous liquid may comprise particles. The particles 616 (e.g.,beads) can be introduced into the channel 602 from a separate channel(not shown in FIG. 6). In some instances, the particles 616 can beintroduced into the channel 602 from a plurality of different channels,and the frequency controlled accordingly. In some instances, differentparticles may be introduced via separate channels. For example, a firstseparate channel can introduce beads and a second separate channel canintroduce biological particles into the channel 602. The first separatechannel introducing the beads may be upstream or downstream of thesecond separate channel introducing the biological particles.

While FIGS. 6A and 6B illustrate one ledge (e.g., step) in the reservoir604, as can be appreciated, there may be a plurality of ledges in thereservoir 604, for example, each having a different cross-sectionheight. For example, where there is a plurality of ledges, therespective cross-section height can increase with each consecutiveledge. Alternatively, the respective cross-section height can decreaseand/or increase in other patterns or profiles (e.g., increase thendecrease then increase again, increase then increase then increase,etc.).

While FIGS. 6A and 6B illustrate the height difference, dh, being abruptat the ledge 608 (e.g., a step increase), the height difference mayincrease gradually (e.g., from about 0 μm to a maximum heightdifference). In some instances, the height difference may decreasegradually (e.g., taper) from a maximum height difference. In someinstances, the height difference may variably increase and/or decreaselinearly or non-linearly. The same may apply to a height difference, ifany, between the first cross-section and the second cross-section.

Example 7

FIGS. 7A and 7B show a cross-section view and a top view, respectively,of another example of a microfluidic device with a geometric feature fordroplet formation. A device 700 can include a channel 702 communicatingat a fluidic connection 706 (or intersection) with a reservoir 704. Insome instances, the device 700 and one or more of its components cancorrespond to the channel 600 and one or more of its components.

An aqueous liquid 712 comprising a plurality of particles 716 may betransported along the channel 702 into the fluidic connection 706 tomeet a second liquid 714 (e.g., oil, etc.) that is immiscible with theaqueous liquid 712 in the reservoir 704 to create droplets 720 of theaqueous liquid 712 flowing into the reservoir 704. At the fluidicconnection 706 where the aqueous liquid 712 and the second liquid 714meet, droplets can form based on factors such as the hydrodynamic forcesat the fluidic connection 706, relative flow rates of the two liquids712, 714, liquid properties, and certain geometric parameters (e.g., Δh,etc.) of the device 700. A plurality of droplets can be collected in thereservoir 704 by continuously injecting the aqueous liquid 712 from thechannel 702 at the fluidic connection 706.

In some instances, the second liquid 714 may not be subjected to and/ordirected to any flow in or out of the reservoir 704. For example, thesecond liquid 714 may be substantially stationary in the reservoir 704.In some instances, the second liquid 714 may be subjected to flow withinthe reservoir 704, but not in or out of the reservoir 704, such as viaapplication of pressure to the reservoir 704 and/or as affected by theincoming flow of the aqueous liquid 712 at the fluidic connection 706.Alternatively, the second liquid 714 may be subjected and/or directed toflow in or out of the reservoir 704. For example, the reservoir 704 canbe a channel directing the second liquid 714 from upstream todownstream, transporting the generated droplets. Alternatively or inaddition, the second liquid 714 in reservoir 704 may be used to sweepformed droplets away from the path of the nascent droplets.

The device 700 at or near the fluidic connection 706 may have certaingeometric features that at least partly determine the sizes and/orshapes of the droplets formed by the device 700. The channel 702 canhave a first cross-section height, h₁, and the reservoir 704 can have asecond cross-section height, h₂. The first cross-section height, h₁, maybe different from the second cross-section height h₂ such that at ornear the fluidic connection 706, there is a height difference of Δh. Thesecond cross-section height, h₂, may be greater than the firstcross-section height, h₁. The reservoir may thereafter graduallyincrease in cross-section height, for example, the more distant it isfrom the fluidic connection 706. In some instances, the cross-sectionheight of the reservoir may increase in accordance with expansion angle,β, at or near the fluidic connection 706. The height difference, Δh,and/or expansion angle, β, can allow the tongue (portion of the aqueousliquid 712 leaving channel 702 at fluidic connection 706 and enteringthe reservoir 704 before droplet formation) to increase in depth andfacilitate decrease in curvature of the intermediately formed droplet.For example, droplet size may decrease with increasing height differenceand/or increasing expansion angle.

While FIGS. 7A and 7B illustrate the height difference, Δh, being abruptat the fluidic connection 706, the height difference may increasegradually (e.g., from about 0 μm to a maximum height difference). Insome instances, the height difference may decrease gradually (e.g.,taper) from a maximum height difference. In some instances, the heightdifference may variably increase and/or decrease linearly ornon-linearly. While FIGS. 7A and 7B illustrate the expanding reservoircross-section height as linear (e.g., constant expansion angle, β), thecross-section height may expand non-linearly. For example, the reservoirmay be defined at least partially by a dome-like (e.g., hemispherical)shape having variable expansion angles. The cross-section height mayexpand in any shape.

Example 8

FIGS. 8A and 8B show a cross-section view and a top view, respectively,of another example of a microfluidic device with a geometric feature fordroplet formation. A device 800 can include a channel 802 communicatingat a fluidic connection 806 (or intersection) with a reservoir 804. Insome instances, the device 800 and one or more of its components cancorrespond to the device 700 and one or more of its components and/orcorrespond to the device 600 and one or more of its components.

An aqueous liquid 812 comprising a plurality of particles 816 may betransported along the channel 802 into the fluidic connection 806 tomeet a second liquid 814 (e.g., oil, etc.) that is immiscible with theaqueous liquid 812 in the reservoir 804 to create droplets 820 of theaqueous liquid 812 flowing into the reservoir 804. At the fluidicconnection 806 where the aqueous liquid 812 and the second liquid 814meet, droplets can form based on factors such as the hydrodynamic forcesat the fluidic connection 806, relative flow rates of the two liquids812, 814, liquid properties, and certain geometric parameters (e.g., Δh,etc.) of the device 800. A plurality of droplets can be collected in thereservoir 804 by continuously injecting the aqueous liquid 812 from thechannel 802 at the fluidic connection 806.

A discrete droplet generated may comprise one or more particles of theplurality of particles 816. As described elsewhere herein, a particlemay be any particle, such as a bead, cell bead, gel bead, biologicalparticle, macromolecular constituents of biological particle, or otherparticles. Alternatively, a discrete droplet generated may not includeany particles.

In some instances, the second liquid 814 may not be subjected to and/ordirected to any flow in or out of the reservoir 804. For example, thesecond liquid 814 may be substantially stationary in the reservoir 804.In some instances, the second liquid 814 may be subjected to flow withinthe reservoir 804, but not in or out of the reservoir 804, such as viaapplication of pressure to the reservoir 804 and/or as affected by theincoming flow of the aqueous liquid 812 at the fluidic connection 806.Alternatively, the second liquid 814 may be subjected and/or directed toflow in or out of the reservoir 804. For example, the reservoir 804 canbe a channel directing the second liquid 814 from upstream todownstream, transporting the generated droplets. Alternatively or inaddition, the second liquid 814 in reservoir 804 may be used to sweepformed droplets away from the path of the nascent droplets.

While FIGS. 8A and 8B illustrate one ledge (e.g., step) in the reservoir804, as can be appreciated, there may be a plurality of ledges in thereservoir 804, for example, each having a different cross-sectionheight. For example, where there is a plurality of ledges, therespective cross-section height can increase with each consecutiveledge. Alternatively, the respective cross-section height can decreaseand/or increase in other patterns or profiles (e.g., increase thendecrease then increase again, increase then increase then increase,etc.).

While FIGS. 8A and 8B illustrate the height difference, Δh, being abruptat the ledge 808, the height difference may increase gradually (e.g.,from about 0 μm to a maximum height difference). In some instances, theheight difference may decrease gradually (e.g., taper) from a maximumheight difference.

In some instances, the height difference may variably increase and/ordecrease linearly or non-linearly. While FIGS. 8A and 8B illustrate theexpanding reservoir cross-section height as linear (e.g., constantexpansion angle), the cross-section height may expand non-linearly. Forexample, the reservoir may be defined at least partially by a dome-like(e.g., hemispherical) shape having variable expansion angles. Thecross-section height may expand in any shape.

Example 9

An example of a device according to the invention is shown in FIGS.9A-9B. The device 900 includes four fluid reservoirs, 904, 905, 906, and907, respectively. Reservoir 904 houses one liquid; reservoirs 905 and906 house another liquid, and reservoir 907 houses continuous phase inthe step region 908. This device 900 include two first channels 902connected to reservoir 905 and reservoir 906 and connected to a shelfregion 920 adjacent a step region 908. As shown, multiple channels 901from reservoir 904 deliver additional liquid to the first channels 902.The liquids from reservoir 904 and reservoir 905 or 906 combine in thefirst channel 902 forming the first liquid that is dispersed into thecontinuous phase as droplets. In certain embodiments, the liquid inreservoir 905 and/or reservoir 906 includes a particle, such as a gelbead. FIG. 9B shows a view of the first channel 902 containing gel beadsintersected by a second channel 901 adjacent to a shelf region 920leading to a step region 908, which contains multiple droplets 916.

Example 10

Variations on shelf regions 1020 are shown in FIGS. 10A-10E. As shown inFIGS. 10A-10B, the width of the shelf region 1020 can increase from thedistal end of a first channel 1002 towards the step region 1008,linearly as in 10A or non-linearly as in 10B. As shown in FIG. 10C,multiple first channels 1002 can branch from a single feed channel 1002and introduce fluid into interconnected shelf regions 1020. As shown inFIG. 10D, the depth of the first channel 1002 may be greater than thedepth of the shelf region 1020 and cut a path through the shelf region1020. As shown in FIG. 10E, the first channel 1002 and shelf region 1020may contain a grooved bottom surface. This device 1000 also includes asecond channel 1002 that intersects the first channel 1002 proximal toits distal end.

Example 11

Continuous phase delivery channels 1102, shown in FIGS. 11A-11D, arevariations on shelf regions 1120 including channels 1102 for delivery(passive or active) of continuous phase behind a nascent droplet. In oneexample in FIG. 11A, the device 1100 includes two channels 1102 thatconnect the reservoir 1304 of the step region 1108 to either side of theshelf region 1120. In another example in FIG. 11 B, four channels 1102provide continuous phase to the shelf region 1120. These channels 1102can be connected to the reservoir 1104 of the step region 1108 or to aseparate source of continuous phase. In a further example in FIG. 11C,the shelf region 1120 includes one or more channels 1102 (white) belowthe depth of the first channel 1102 (black) that connect to thereservoir 1104 of the step region 1108. The shelf region 1120 containsislands 1122 in black. In another example FIG. 11D, the shelf region1120 of FIG. 11C includes two additional channels 1102 for delivery ofcontinuous phase on either side of the shelf region 1120.

Example 12

An embodiment of a device according to the invention is shown in FIG.12. This device 1200 includes two channels 1201, 1202 that intersectupstream of a droplet formation region. The droplet formation regionincludes both a shelf region 1220 and a step region 1208 disposedbetween the distal end of the first channel 1201 and the step region1208 that lead to a collection reservoir 1204. The black and whitearrows show the flow of liquids through each of first channel 1201 andsecond channel 1202, respectively. In certain embodiments, the liquidflowing through the first channel 1201 or second channel 1202 includes aparticle, such as a gel bead. As shown in the FIG. 12, the width of theshelf region 1220 can increase from the distal end of a first channel1201 towards the step region 1208; in particular, the width of the shelfregion 1220 in FIG. 12 increases non-linearly. In this embodiment, theshelf region extends from the edge of a reservoir to allow dropletformation away from the edge. Such a geometry allows droplets to moveaway from the droplet formation region due to differential densitybetween the continuous and dispersed phase.

Example 13

An embodiment of a device according to the invention for multiplexeddroplet formation is shown in FIGS. 13A-13B. This device 1300 includesfour fluid reservoirs, 1304, 1305, 1306, and 1307, and the overalldirection of flow within the device 1300 is shown by the black arrow inFIG. 13A. Reservoir 1304 and reservoir 1306 house one liquid; reservoir1305 houses another liquid, and reservoir 1307 houses continuous phaseand is a collection reservoir. Fluid channels 1301, 1303 directlyconnect reservoir 1304 and reservoir 1306, respectively, to reservoir1307; thus, there are four droplet formation region in this device 1300.Each droplet formation region has a shelf region 1320 and a step region1308. This device 1300 further has two channels 1302 from the reservoir1305 where each of these channels splits into two separate channels attheir distal ends. Each of the branches of the split channel intersectsthe first channels 1301 or 1303 upstream of their connection to thecollection reservoir 1307. As shown in the zoomed in view of the dottedline box in FIG. 13B, second channel 1302, with its flow indicated bythe white arrow, has its distal end intersecting a channel 1302 fromreservoir 1304, with the flow of the channel indicated by the blackarrow, upstream of the droplet formation region. The liquid fromreservoir 1304 and reservoir 1306, separately, are introduced intochannels 1301, 1303 and flow towards the collection reservoir 1307. Theliquid from the second reservoir 1305 combines with the fluid fromreservoir 1304 or reservoir 1306, and the combined fluid is dispersedinto the droplet formation region and to the continuous phase. Incertain embodiments, the liquid flowing through the first channel 1301or 1303 or second channel 1302 includes a particle, such as a gel bead.

Example 14

Examples of devices according to the invention that include two dropletformation regions are shown in FIGS. 14A-14B. The device 1400 of FIG.14A includes three reservoirs, 1405, 1406, and 1407, and the device 1400of FIG. 14B includes four reservoirs, 1404, 1405, 1406, and 1407. Forthe device 1400 of FIG. 14A, reservoir 1405 houses a portion of thefirst fluid, reservoir 1406 houses a different portion of the firstfluid, and reservoir 1407 houses continuous phase and is a collectionreservoir. In the device 1400 of FIG. 14B, reservoir 1404 houses aportion of the first fluid, reservoir 1405 and reservoir 1406 housedifferent portions of the first fluid, and reservoir 1407 housescontinuous phase and is a collection reservoir. In both devices 1400,there are two droplet formation regions. For the device 1400 of FIG.14A, the connections to the collection reservoir 1407 are from thereservoir 1406, and the distal ends of the channels 1401 from reservoir1405 intersect the channels 1402 from reservoir 1406 upstream of thedroplet formation region. The liquids from reservoir 1405 and reservoir1406 combine in the channels 1402 from reservoir 1406, forming the firstliquid that is dispersed into the continuous phase in the collectionreservoir 1407 as droplets. In certain embodiments, the liquid inreservoir 1405 and/or reservoir 1406 includes a particle, such as a gelbead.

In the device 1400 of FIG. 14B, each of reservoir 1405 and reservoir1406 are connected to the collection reservoir 1407. Reservoir 1404 hasthree channels 1401, two of which have distal ends that intersect eachof the channels 1402, 1403 from reservoir 1404 and reservoir 1406,respectively, upstream of the droplet formation region. The thirdchannel 1401 from reservoir 1404 splits into two separate distal ends,with one end intersecting the channel 1402 from reservoir 1405 and theother distal end intersecting the channel 1403 from reservoir 1406, bothupstream of droplet formation regions. The liquid from reservoir 1404combines with the liquids from reservoir 1405 and reservoir 1406 in thechannels 1402 from reservoir 1405 and reservoir 1406, forming the firstliquid that is dispersed into the continuous phase in the collectionreservoir 1407 as droplets. In certain embodiments, the liquid inreservoir 1404, reservoir 1405, and/or reservoir 1406 includes aparticle, such as a gel bead.

Example 15

An embodiment of a device according to the invention that has fourdroplet formation regions is shown in FIG. 15. The device 1500 of FIG.15 includes four reservoirs, 1504, 1505, 1506, and 1507; the reservoirlabeled 1504 is unused in this embodiment. In the device 1500 of FIG.15, reservoir 1505 houses a portion of the first fluid, reservoir 1506houses a different portion of the first fluid, and reservoir 1507 housescontinuous phase and is a collection reservoir. Reservoir 1506 has fourchannels 1502 that connect to the collection reservoir 1507 at fourdroplet formation regions. The channels 1502 from originating atreservoir 1506 include two outer channels 1502 and two inner channels1502. Reservoir 1505 has two channels 1501 that intersect the two outerchannels 1502 from reservoir 1506 upstream of the droplet formationregions. Channels 1501 and the inner channels 1502 are connected by twochannels 1503 that traverse, but do not intersect, the fluid paths ofthe two outer channels 1502. These connecting channels 1503 fromchannels 1501 pass over the outer channels 1502 and intersect the innerchannels 1502 upstream of the droplet formation regions. The liquidsfrom reservoir 1505 and reservoir 1506 combine in the channels 1502,forming the first liquid that is dispersed into the continuous phase inthe collection reservoir 1507 as droplets. In certain embodiments, theliquid in reservoir 1505 and/or reservoir 1506 includes a particle, suchas a gel bead.

Example 16

An embodiment of a device according to the invention that has aplurality of droplet formation regions is shown in FIGS. 16A-16B (FIG.16B is a zoomed in view of FIG. 16A), with the droplet formation regionincluding a shelf region 1620 and a step region 1608. This device 1600includes two channels 1601, 1602 that meet at the shelf region 1620. Asshown, after the two channels 1601, 1602 meet at the shelf region 1620,the combination of liquids is divided, in this example, by four shelfregions. In certain embodiments, the liquid with flow indicated by theblack arrow includes a particle, such as a gel bead, and the liquid flowfrom the other channel, indicated by the white arrow, can move theparticles into the shelf regions such that each particle can beintroduced into a droplet.

Example 17

An embodiment of a method of modifying the surface of a device using acoating agent is shown in FIGS. 17A-17B. In this example, the surface ofthe droplet formation region of the device 1700, e.g., the rectangulararea connected to the circular shaped collection reservoir 1704, iscoated with a coating agent 1722 to modify its surface properties. Tolocalize the coating agent to only the regions of interest, the firstchannel 1701 and second channel 1702 of the device 1700 are filled witha blocking liquid 1724 (Step 2 of FIG. 17A) such that the coating agent1722 cannot contact the channels 1701, 1702. The device 1700 is thenfilled with the coating agent 1722 to fill the droplet formation regionand the collection reservoir 1704 (Step 3 of FIG. 17A). After thecoating process is complete, the device 1700 is flushed (Step 4 of FIG.17A) to remove both the blocking liquid 1724 from the channels and thecoating agent 1722 from the droplet formation region and the collectionreservoir 1704. This leaves behind a layer of the coating agent 1722only in the regions where it is desired. This is further exemplified inthe micrograph of FIG. 17B, the blocking liquid (dark gray) fills thefirst channel 1701 and second channel 1702, preventing ingress of thecoating agent 1722 (white) into either the first channel 1701 or thesecond channel 1702 while completely coating the droplet formationregion and the collection reservoir 1704. In this example, the firstchannel 1701 is also acting as a feed channel for the blocking liquid1724, shown by the arrow for flow direction in FIG. 17B.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. It is not intendedthat the invention be limited by the specific examples provided withinthe specification. While the invention has been described with referenceto the aforementioned specification, the descriptions and illustrationsof the embodiments herein are not meant to be construed in a limitingsense. Numerous variations, changes, and substitutions will now occur tothose skilled in the art without departing from the invention.Furthermore, it shall be understood that all aspects of the inventionare not limited to the specific depictions, configurations or relativeproportions set forth herein which depend upon a variety of conditionsand variables. It should be understood that various alternatives to theembodiments of the invention described herein may be employed inpracticing the invention. It is therefore contemplated that theinvention shall also cover any such alternatives, modifications,variations or equivalents. It is intended that the following claimsdefine the scope of the invention and that methods and structures withinthe scope of these claims and their equivalents be covered thereby.

Other embodiments are in the claims.

What is claimed is:
 1. A method of producing droplets comprising: a)providing a device comprising: i) a first channel having a firstproximal end, a first distal end, a first depth, and a first width, thefirst channel comprising particles and a first liquid; and ii) a dropletformation region comprising a second liquid and being in fluidcommunication with the first channel, wherein the droplet formationregion has a width greater than the first width or a depth greater thanthe first depth; wherein the first liquid is immiscible with the secondliquid; and b) flowing the first liquid from the first channel to thedroplet formation region to produce droplets of the first liquid in thedroplet formation region and one or more particles, wherein the dropletsare dispersed in the second liquid, and wherein the device is capable offorming droplets with the second liquid flowing in response todisplacement by the droplets being formed; and c) transporting thedroplets from the droplet formation region by buoyancy.
 2. The method ofclaim 1, wherein the device further comprises a collection region, andthe droplets are substantially stationary in the collection region. 3.The method of claim 1, wherein the first depth decreases in theproximal-to-distal direction in at least a portion of the first channel.4. The method of claim 1, wherein the first depth increases in theproximal-to-distal direction in at least a portion of the first channel.5. The method of claim 1, further comprising a first reservoir in fluidcommunication with the first proximal end.
 6. The method of claim 5,wherein the first reservoir further comprises the particles.
 7. Themethod of claim 1, wherein step b) produces droplets having a singleparticle.
 8. The method of claim 1, wherein the particles have about thesame density as the first liquid.
 9. The method of claim 1, wherein thefirst liquid is aqueous or miscible with water.
 10. The method of claim1, wherein the density of the first liquid is lower than the density ofthe second liquid.
 11. The method of claim 1, wherein the density of thefirst liquid is higher than the density of the second liquid.
 12. Themethod of claim 1, wherein the device further comprises a second channelhaving a second proximal end, a second distal end, a second depth, and asecond width; wherein the second channel intersects the first channelbetween the first proximal end and the first distal end, the secondchannel comprising a third liquid; wherein the third liquid combineswith the first liquid at the intersection, and the droplets furthercomprise the third liquid.
 13. The method of claim 12, wherein thesecond depth decreases in the proximal-to-distal direction in at least aportion of the second channel.
 14. The method of claim 12, wherein thesecond depth increases in the proximal-to-distal direction in at least aportion of the second channel.
 15. The method of claim 12, wherein thethird liquid is aqueous or miscible with water.
 16. The method of claim12, wherein the density of the third liquid is lower than the density ofthe second liquid.
 17. The method of claim 12, wherein the density ofthe third liquid is higher than the density of the second liquid. 18.The method of claim 12, wherein the device further comprises a secondreservoir in fluid communication with the second proximal end.
 19. Themethod of claim 1, wherein the droplet formation region comprises ashelf region having a third depth and a third width, wherein the shelfregion has at least one inlet and at least one outlet.
 20. The method ofclaim 19, wherein the third width increases from the inlet of the shelfregion to the outlet of the shelf region.
 21. The method of claim 1,wherein the droplet formation region comprises a step region having afourth depth and a fourth width.
 22. The method of claim 21, wherein thedroplet formation region further comprises a shelf region that isdisposed between the first distal end and the step region.
 23. Themethod of claim 22, wherein the device further comprises a third channelhaving a third proximal end and a third distal end, wherein the thirdproximal end is in fluid communication with the shelf region, andwherein the third distal end is in fluid communication with the stepregion.
 24. The method of claim 1, wherein the droplet formation regioncomprises a plurality of inlets in fluid communication with the firstproximal end and a plurality of outlets.
 25. The method of claim 24,wherein the number of inlets and the number of outlets is the same. 26.The method of claim 1, wherein the depth of the droplet formation regionis at least 100 μm.
 27. The method of claim 1, wherein the droplets havea volume less than 1000 pL.